Exp. Eye Res. (1999) 68, 117–127Article No. exer.1998.0606, available online at http:}}www.idealibrary.com on

Protection from Oxidative Insult in Glutathione Depleted Lens

Epithelial Cells

JOHN R. REDDANa*, FRANK J. GIBLINb, RAZAN KADRYa, VICTOR R. LEVERENZb,

JOHN T. PENAa and DOROTHY C. DZIEDZICa

aDepartment of Biological Sciences and bEye Research Institute, Oakland University, Rochester,

MI 48309-4476, U.S.A.

(Received Oxford 4 September 1998 and accepted in revised from 26 October 1998)

It has previously been shown that TEMPOL, n-propyl gallate and deferoxamine, compounds that limit theavailability of Fe+# and prevent the generation of hydroxyl radicals, protect cultured rabbit lens epithelialcells from H

#O#-induced damage. In view of the importance of glutathione as an antioxidant and the

decrease in GSH that is known to accompany most forms of cataract, we investigated whether thesecompounds protected cultured lens epithelial cells from H

#O#when the cells were artificially depleted of

glutathione. Treatment of lens epithelial cells with 1-chloro-2,4-dinitrobenzene (CDNB), a compound thatirreversibly binds to glutathione, or buthionine sulfoximine (BSO), an inhibitor of glutathionebiosynthesis, reduced the glutathione content to an average of 15–20% of the control values without aconcomitant increase in oxidized glutathione. Morphological changes were assessed by phase contrastand electron microscopy. In order to assess growth, cells in 5 ml serum-free MEM were exposed to aninitial concentration of 0±05 m H

#O#(for 50,000 cells) or 2 doses of 0±5 m H

#O#(for 800,000 cells).

After exposure to H#O#, medium was replaced with MEM plus 8% rabbit serum; cells were fed on days 3

and 6 and counted on day 7.When 50,000 or 800,000 cells with decreased glutathione were exposed to 0±05 or 0±5 m H

#O#the

H#O#

was cytotoxic, whereas cells treated with H#O#

alone remained viable but showed inhibitedproliferation. An unexpected finding was that cells continued to remove H

#O#from the medium at normal

rates even when the GSH level was reduced. Cells treated with CDNB or BSO alone exhibitedmorphological and growth properties comparable to untreated cells. Cells treated with CDNB or BSO andthen with H

#O#exhibited decreased cell-to-cell contact, nuclear shrinkage, and arborization when viewed

with phase-contrast microscopy and showed extensive nuclear and cytoplasmic degeneration at the EMlevel. Cell death was determined by dye exclusion and confirmed by video microscopy. When cells weretreated with CDNB or BSO and subsequently treated with TEMPOL, n-propyl gallate or deferoxamine andthen challenged with H

#O#

cytotoxicity was prevented and the cells were capable of growth. The datashow that H

#O#was not lethal to glutathione-depleted lens epithelial cells when they were treated with

compounds that prevented the generation of reactive oxygen species. In addition, the results indicate thatGSH has an important protective role independent of its ability to decompose H

#O#

via glutathioneperoxidase. # 1999 Academic Press

Key words : cultured rabbit lens epithelial cells ; cell line N}N1003A; buthionine sulfoximine; CDNB;glutathione; hydrogen peroxide ; oxidative stress ; deferoxamine; TEMPOL; n-propyl gallate.

1. Introduction

The lens epithelium plays an important role for

transport in the whole lens (Delamere et al., 1996),

provides the cells that differentiate into lens fibers

(Reddan, 1982), and contains systems that are capable

of protecting the lens from oxidative insult (Reddy,

1971; Giblin et al., 1985; Nagaraj et al., 1991;

Spector, 1991; Reddan et al., 1993). The lens

epithelium is known to have the highest concentration

of GSH in the lens (Reddy, 1971). Among other

functions, GSH acts as an antioxidant, detoxifies

xenobiotics and has been reported to decrease the

reactions of lens proteins with sugars, thus limiting

*Address correspondence to: John R. Reddan, Department ofBiological Sciences, Oakland University, Rochester, M1 48309-4476, U.S.A.

damaging glycation and carbamylation reactions

(Packer and Cadenas, 1995; Harding, Blakytny and

Ganea, 1996). The importance of GSH stems from the

finding that the tripeptide is decreased in most forms of

cataracts (Reddy, 1974; Harding, Blakytny and

Ganea, 1996). In humans, it has been reported that a

decrease in GSH precedes irreversible cataract forma-

tion (Rathbun, Schmidt and Holleschau, 1993). The

synthesis of reduced glutathione decreases with age in

the lens and it has been reported that the transport of

-cyst(e)ine into the lens is the rate-limiting factor in

GSH synthesis in the intact human lens (Rathbun and

Murray, 1991). Such transport must involve the lens

epithelium.

The developing lens is more sensitive to -buthio-

nine sulfoximine (BSO)-induced depletion of GSH than

any other organ (Martensson et al., 1989). Near total

depletion of glutathione by in vitro administration of

BSO, a specific inhibitor of GSH biosynthesis induces

0014–4835}99}010117­11 $30.00}0 # 1999 Academic Press

118 J. R. REDDAN ET AL.

cataracts in 9–12 day old mice (Calvin, Medvedovsky

and Worgul, 1986). Early epithelial lesions accom-

pany cataract formation in BSO treated mice (Laver et

al., 1993). The fibers are swollen, mitotic activity

increases, the orderly arrangement of the meridional

rows is disrupted (Calvin et al., 1991), electrolyte

imbalance occurs (Calvin et al., 1992b), and crystal-

lins are modified (Calvin et al., 1992a). BSO-induced

cataracts in newborn rats or mice are prevented by the

antioxidant alpha lipoic acid (Maitra et al., 1995), are

prevented or delayed by the administration of GSH

monoester (Martensson et al., 1989), and are delayed

or prevented in newborn rats by ascorbate (Martens-

son and Meister, 1991). Thiols or ascorbate prevent

the progression of BSO-induced cataracts in cul-

tured mouse lenses (Calvin et al., 1997b). Calvin

et al. reported that H7, an inhibitor of serine-

threonine protein kinase prevents BSO-induced

cataracts in mouse lenses (Calvin, Banerjee and Fu,

1997a).

We have demonstrated the importance of the

glutathione redox cycle in protecting lens epithelial

cells from low levels of H#O#(Giblin et al., 1985) and

defined the relative roles of the glutathione redox cycle

and catalase in detoxifying H#O#(Giblin et al., 1990).

The maximal levels of H#O#present in human aqueous

humor may be lower than previously reported

(Sharma et al., 1997; Spector, Ma and Wang,

1998). Recent studies show that fresh bovine aqueous

humor is capable of generating H#O#, a compound

that can produce damaging oxygen species (Spector

et al., 1998). Lens epithelials cells are particularly

vulnerable to low maintained levels of H#O#

if gluta-

thione reductase, a key component of the glutathione

redox cycle, is inhibited (Ikebe et al., 1989).

Cultured lens epithelial cells from older rabbits

with a lower activity of glutathione reductase are

more susceptible to H#O#

cytotoxicity than cells from

younger animals with a high level of the enzyme

(Reddan et al., 1988). Glutathione reductase is

reportedly inactive in the epithelium of a significant

number of human cataract patients (Horwitz et al.,

1987).

We have shown that the nitroxide free radical,

TEMPOL, n-propyl gallate (nPG) and deferoxamine,

compounds that prevent the generation of reactive

oxygen species, protect cultured lens epithelial cells

with normal levels of GSH from H#O#-induced damage

(Reddan et al., 1992, 1993, 1998). Here, we investi-

gated whether TEMPOL, deferoxamine or n-propyl

gallate could protect epithelial cells from H#O#

when

the endogenous level of intracellular glutathione was

depleted with either BSO or 1-chloro-2,4-dinitro-

benzene (CDNB). BSO is a specific inhibitor of gamma

glutamyl cysteine synthetase (Griffith, 1982), the

enzyme that catalyzes the initial step of glutathione

synthesis. CDNB conjugates glutathione through a

reaction catalyzed by glutathione-S-transferase (Reddy

et al., 1988).

2. Materials and Methods

Culture of LECs

Cells (line N}N1003A, Reddan et al., 1986) at

population doubling levels 25–75, were grown in

Eagle’s MEM with Earle’s salts, supplemented with 8%

rabbit serum (Sigma, St. Louis, MO, U.S.A.) and 50 µg

ml−" gentamicin (Sigma). The cells retain the tran-

scription factors required for activating the murine

αA- and γ-crystallin gene promoters (Reddan et al.,

1986; Meakin et al., 1989) and express pax 6, a

master gene involved in eye development (Krausz et

al., 1996). Experiments were started 6 days after cells

were confluent. Cells were enzymatically removed

from the stock plates 1 day before the experiments,

counted with a Coulter Counter (Coulter Electronics,

Miami, FL, U.S.A.), and 50,000 or 800,000 cells were

plated into 60 mm culture plates containing 5 ml of

MEM plus 1% rabbit serum. Cells cultured in 1%

rabbit serum in MEM remain viable but usually do not

grow. Sixteen to 20 hr later, cells from 3 plates were

counted to determine the actual starting number. Cells

from plates that were not counted were cultured for

30 minutes in serum-free MEM to minimize the

interaction of H#O#

or other test compounds with

serum proteins.

Measurement of GSH and GSSG

Levels of GSH and GSSG in 800,000 cells were

measured with an electrochemical detector (Bio-

analytical Systems, Inc., West Lafayette, IN, U.S.A.)

which is sensitive to 5 pmol of either GSH or GSSG

(Chakrapani et al., 1990). Concentrations of GSH as

non-protein sulfhydryls were also measured spectro-

photometrically (Giblin et al., 1985). The levels of GSH

and GSSG in 50,000 cells were measured using a

slight modification of the techniques of Tietze (Tietze,

1969) and Griffith (Griffith, 1980).

Depletion of Glutathione

The dose of BSO (Sigma) required to lower the

glutathione level by approximately 85% was de-

termined by culturing 800,000 cells overnight in 1%

rabbit serum in MEM containing 0±0 to 1±0 m BSO.

At 20 hr, cells were counted and glutathione was

determined. The influence of CDNB on GSH levels was

determined by pre-treating 800,000 cells with 0±02 or

0±1 m CDNB for 10–60 minutes.

For BSO treatment, 50,000 or 800,000 cells were

seeded into dishes containing 0±5 m BSO in MEM

with 1% rabbit serum. Three plates were counted 20

hr later to reaffirm the starting count, and the

remaining plates were cultured in serum-free medium

as above for 30 minutes. For CDNB (Sigma) treatment,

cells were cultured in serum-free MEM containing

0±02 m CDNB for 10 minutes. The medium was then

PROTECTION IN GSH DEPLETED CELLS 119

replaced with either MEM for controls or with an

antioxidant and}or H#O#.

Antioxidant Treatment

Cells cultured in nPG (Sigma) and deferoxamine

(Sigma) were pre-treated only. nPG (1 m) in serum-

free MEM was added to the plates for 10 minutes

and then replaced with serum-free MEM or with H#O#

in serum-free MEM. Deferoxamine was a 60 minute

pre-treatment at 20 m in serum-free MEM. TEMPOL

(Sigma, 5 m) was present with the bolus of H#O#(co-

treatment). These doses of deferoxamine, nPG or

TEMPOL protect cultured rabbit lens epithelial cells

with normal glutathione levels from H#O#

insult

(Reddan et al., 1992, 1993, 1998).

Hydrogen Peroxide Treatment

A 30% solution of H#O#

(Sigma) was diluted to 25

m with deionized water and added to MEM to give

the desired concentration. H#O#

in the MEM was

measured at various times with a YSI model 23A

analyzer equipped with a selective H#O#

electrode

(Yellow Springs Instrument Company, Yellow Springs,

OH, U.S.A.). One bolus of 0±05 m (final concen-

tration) H#O#in MEM was added to 50,000 cells. Three

hours later, the medium was replaced with MEM

containing 8% rabbit serum. For 800,000 cells, 2

separate 3-hr treatments of 0±5 m H#O#were given to

the cells. The amount of H#O#-containing medium

added was adjusted for the actual cell number to an

equivalent of 5 ml}800,000 or 5 ml}50,000 cells. At

the end of the H#O#

treatment the medium was

replaced and cells were cultured in MEM containing

8% rabbit serum.

Growth Experiments

For growth experiments, the serum-containing

medium was replaced on days 3 and 6 and cells were

counted on day 7 using a Coulter Counter.

Cell Viability and Morphology

Cell viability was evaluated using Trypan blue

exclusion. At least 500 cells}plate were counted (n¯4). In addition, cell behavior was monitored with a

Panasonic time lapse video cassette recorder and

Hitachi color video camera connected to an inverted

phase-contrast microscope as previously described

(Reddan et al., 1993). 50,000 or 800,000 cells were

treated with 0±5 m BSO for 20 hr and rinsed for 30

min in serum-free MEM. At this time, the cells were

given a 10 min treatment of 0±0 or 1 m nPG, and

then the medium was changed to serum-free MEM

with 0±05 m H#O#. Morphology was monitored for

the entire 6 hr treatment with H#O#. The influence of

H#O#

on cell death and cell growth was monitored

throughout a 7 day culture period using a closed-

circuit TV monitor. Sealed flasks of cells remained on

the time-lapse set-up for 24 hr and were then replaced

with other flasks which had been cultured in a

standard CO#

incubator (Reddan et al., 1986).

Transmission Electron Microscopy

Cells in 60 mm plates (Corning) were treated as

described above and rinsed with 5 ml of MEM. Cells

were fixed in 2±5% glutaraldehyde prepared in 0±1

cacodylate buffer for 1±5 hr at 22 °C. After rinsing

twice with 0±1 cacodylate buffer at 22 °C, the cells

were post-fixed in 0±5% OsO%at 4 °C for 50 minutes.

The cells were briefly rinsed in 0±1 cacodylate buffer

at 4 °C. The rinse was replaced and the cells remained

in 5 ml of 0±1 cacodylate buffer for 24 hr. The cells

were then dehydrated through an ascending series of

ethanol, infiltrated with EPON 812 for 4 days and

polymerized overnight at 45 °C. Ultra-thin sections

were cut, mounted on nickel grids, post-stained with

2% uranyl acetate and lead citrate. En face sections

were examined and photographed on a Phillips 410

transmission electron microscope.

3. Results

The effect of BSO and CDNB on GSH levels in

800,000 or 50,000 lens epithelial cells is shown in

Table I. Untreated cells had a GSH content of 33³6

nmol}800,000 cells and 3±6³1±5 nmol}50,000 cells.

A 20 hr treatment of cultured cells with 0±5 or 1±0 m

BSO or a 10 minute treatment of 800,000 cells with

0±02 m CDNB brought about an 80–85% reduction

in GSH (Table I). Higher doses of CDNB were toxic.

Comparable GSH values in BSO and CDNB treated cells

were obtained using spectrophotometric or electro-

chemical methods. Oxidized glutathione was not

detectable in BSO}H#O#or CDNB}H

#O#-treated cells as

determined with the electrochemical detector or the

Tietze assay. It was conceivable that treatment with

CDNB or BSO would influence the ability of 800,000

or 50,000 cells to remove H#O#

from the culture

medium. However, pre-treatment with CDNB [Fig.

1(A)] or BSO (data not shown) did not affect the rate

at which the 800,000 cells removed peroxide. The

blank that contained no cells showed a relatively

stable level of H#O#

in the medium. Treatment of

50,000 cells with either BSO [Fig. 1(B)] or CDNB (data

not shown) also did not affect the rate of removal of

H#O#from the culture medium. The results shown in

Figs 1(A) and (B) are virtually identical for either BSO

or CDNB.

Cells with lowered levels of GSH were either

challenged with 0±05 m H#O#

for 50,000 cells, or

with 2 doses of 0±5 m H#O#

for 800,000 cells. Cells

with a normal GSH level treated with 2 doses of H#O#

did not exhibit damage at 6 hr (data not shown). In

addition, cells with lowered GSH levels were either

120 J. R. REDDAN ET AL.

T I

Effect of CDNB or BSO on GSH levels in cultured rabbit lens epithelial cells

TreatmentGSH, nmol}

800,000 cells % of controlGSH, nmol}50,000 cells % of control

MEM (control) 33³6 100³18 3±6³1±5 100³41CDNB 5³2 14³6 0±7³0±5 20³14BSO 5³2 16³6 0±7³0±5 19³15

800,000 or 50,000 rabbit lens epithelial cells were cultured in MEM containing 1% rabbit serum for 20 hr and in serum-free MEM (control)for 30 minutes. For the CDNB treatment, cells were cultured for an additional 10 minutes in 0±02 m CDNB immediately after culture in serum-free MEM. For BSO experiments, the cells were cultured in MEM containing 1% rabbit serum and 0±5 m BSO for 20 hr and for 30 minutesin serum-free MEM. The concentration of GSH was measured 10 minutes after exposure to CDNB or after the BSO treated cells were in serum-free MEM for 30 minutes. Levels of oxidized glutathione were undetectable in the cells under all conditions. (means³.., n¯12)

F. 1 : Lowered GSH did not affect the rate of removal ofH

#O#

by the cultured rabbit lens epithelial cells. (A) Cells(800,000) were pre-treated with CDNB and then exposed toH

#O#(_), or treated with H

#O#alone (E). Blanks consisted

of medium alone without cells, MEM­H#O#

(D) ; CDNB­MEM­H

#O#

(^). (B) 50,000 cells were pretreated withBSO and then exposed to H

#O#

(U) or treated with H#O#

alone (E). The blank was MEM­H#O#

(D). (means³..,n¯4).

treated with 20 m deferoxamine for 1 hr or with 1

m nPG for 10 minutes and then exposed to H#O#.

Other cells were co-treated with 5 m TEMPOL and

H#O#. When 800,000 cells were treated with CDNB or

BSO and then subjected to 0±5 m H#O#and examined

6 hr later with the phase contrast microscope, the cells

were dead [Fig. 2(A)]. They exhibited shriveled nuclei.

The cytoplasm was not homogenous and exhibited

cytoskeletal remains. Cells that were treated with

BSO}nPG}H#O#

remained viable and had a typical

epithelial morphology [Fig. 2(B)]. Identical results

were obtained at 6 hr with 50,000 cells (data not

shown). It should be noted that the results obtained

with nPG were identical to those obtained with either

TEMPOL or deferoxamine.

Cells viability or death was also determined by

Trypan blue exclusion in 800,000 cells treated with

H#O#BSO}H

#O#or BSO}nPG}H

#O#. Cells treated with

H#O#or BSO}nPG}H

#O#at 6 hr exhibited viabilities of

97 and 98%, respectively. These results paralleled

those obtained in cells treated with TEMPOL and

deferoxamine. However, cells treated with BSO}H#O#

exhibited a viability of less than 1%. Time-lapse video

microscopy confirmed the death found in BSO}H#O#-

treated cells and the protection afforded by each of the

antioxidants.

To prove that the protection afforded by the

antioxidants was not merely a short-term effect,

800,000 cells were cultured for 7 days following

exposure to H#O#. At 7 days, untreated cells [Fig. 3(A)]

and cells with high GSH levels exposed to H#O#

[Fig.

3(B)] exhibited a normal morphology. In contrast, cells

treated with CDNB or BSO and then exposed to H#O#

showed extensive damage [Fig. 3(C)]. The damage and

death were prevented by nPG [Fig. 3(D)], deferoxamine

or TEMPOL (results not shown).

The extent of morphological damage 7 days after

BSO}H#O#-treatment and the protection afforded by

the antioxidants were examined with the transmission

electron microscope. Cells treated with BSO}H#O#

exhibited extensive nuclear and cytoplasmic degener-

ation [Fig. 4(A)]. In many cells, the mitochondria ap-

peared swollen, the nuclear material was clumped, and

the nuclearmembrane appeared to be disrupted.Micro-

filaments were visible in portions of the damaged cells.

Cells treated with BSO}nPG}H#O#[Fig. 4(B)] were not

PROTECTION IN GSH DEPLETED CELLS 121

F. 2. Photomicrographs of cultured rabbit lens epithelial cells. 800,000 cells were cultured for 20 hr in MEM containing1% rabbit serum and BSO. Photographs were taken after 2 separate 3 hr treatments with 0±5 m H

#O#following pretreatment

with (A) BSO, (B) BSO}nPG. Note the protective effect of nPG. (¬200)

F. 3. Photomicrographs of cultured rabbit lens epithelial cells. 800,000 cells were cultured in (A) MEM or in MEM plus H#O#

following (B) no pretreatment, (C) CDNB pretreatment, or (D) CDNB}nPG pretreatment. Six hours later, cells were cultured inMEM containing 8% rabbit serum and were photographed after 7 days of culture. Note the protection afforded by nPG. (¬127)

damaged. Fine structural studies were not done with

TEMPOL or deferoxamine in cells with lowered GSH

levels since there was no evidence of damage at the

light microscope level.

Since mitosis is a sensitive indicator of oxidative

damage, we investigated the effect of H#O#

and of

antioxidants and H#O#

on cell proliferation in GSH

depleted and non-GSH depleted lens epithelial cells. As

122 J. R. REDDAN ET AL.

F. 4. Transmission electron photomicrographs of cultured rabbit lens epithelial cells. (A) 800,000 cells were treatedovernight with 0±5 m BSO, exposed to H

#O#and fixed 7 days later. Note the extensive damage (¬5340). (B) Cells were treated

with BSO as in A and then with nPG for 10 minutes and exposed to 0±5 m H#O#(¬5340). N¯nucleus, M¯mitochondria ;

D¯nuclear debris ; SD¯ small dense bodies ; MF¯microfilaments.

F. 5. Propyl gallate overcomes H#O#-induced growth inhibition in glutathione-depleted cells. In some cells GSH levels were

lowered by treatment with CDNB (solid bars). Cells (800,000) were then exposed to 0±0 or 0±5 m H#O#

and grown inMEM­8% rabbit serum for 7 days. Note that nPG prevented cell death and permitted cell growth in H

#O#

and CDNB}H#O#-

treated cells (far right). Pretreatment with CDNB is shown by solid bars ; pretreatment with MEM alone is shown by open bars.Starting count is indicated by the arrow. (means³.., n¯8)

shown in Fig. 5, when 800,000 cells were cultured in

MEM containing 8% rabbit serum with or without

pretreatment with CDNB, cell number increased to

1±3¬10' which is less than a 2 fold increase above the

starting count. When cells were treated with CDNB}-

nPG, growth was comparable to controls. When cells

PROTECTION IN GSH DEPLETED CELLS 123

F. 6. Propyl gallate overcomes H#O#-induced growth inhibition in glutathione-depleted cells. GSH levels were lowered by

treatment with CDNB. Cells (50,000) were exposed to 0±0 or 0±05 m H#O#and then grown in MEM­8% rabbit serum. Note

that nPG prevented cell death and permitted growth in H#O#

and CDNB}H#O#-treated cells (far right). Open bars : MEM

pretreatment, solid bars : CDNB pretreatment. Starting count is indicated by the arrow. (means³.., n¯6)

were exposed to H#O#the number did not increase. A

review of time lapse video microscopy throughout the

7 day period after H#O#treatment revealed that almost

all of the cells remained in a non-dividing state.

Although cell death and mitosis were observed in

H#O#-treated cells, these events occurred very in-

frequently. In CDNB}H#O#

treated cells, the counts

decreased to approximately 300,000. The cells re-

maining on the culture plates were extensively

damaged. Although the cells were not viable, they

would still be counted on the Coulter counter which

does not differentiate between live and dead cells. The

finding that we wish to emphasize is that treatment

with any of the antioxidants, i.e., nPG, TEMPOL, or

deferoxamine, in BSO or CDNB treated cells (i.e., those

cells with low GSH levels) prevented the H#O#-induced

inhibition of cell division and cell death. Since the data

for all antioxidants were virtually identical, only the

CDNB}nPG}H#O#

results are shown (Fig. 5, far right

bar). The generalization that all three antioxidants

protected either BSO or CDNB-treated cells applies to

all of the present studies.

As shown in Fig. 5, when 800,000 cells were

treated with CDBN}nPGLH#O#, there was less than a

two-fold increase in growth after 7 days. The 800,000

cells most likely showed limited growth due to contact

inhibition. It was possible that the cells treated with

antioxidants and BSO}H#O#

or CDNB}H#O#

might be

capable of completing only one round of cell division.

Therefore, experiments were initiated with fewer cells

to provide space for more substantial growth. In these

experiments, 50,000 cells were exposed to 0±05 m

H#O#. The morphological damage caused by H

#O#

in

50,000 GSH-depleted cells and the protective effect of

the antioxidants paralleled those noted in experiments

with 800,000 cells. When 50,000 cells were cultured

in MEM containing 8% rabbit serum and counted 7

days later, the cells showed a 24-fold increase in

number (Fig. 6). It should be noted that treatment

with nPG, TEMPOL or deferoxamine alone did not

curtail cell growth (Fig. 6). Cells exposed to 0±05 m

H#O#

alone exhibited little growth (Fig. 6) and were

enlarged whereas those treated with either BSO}H#O#

or CDNB}H#O#were moribund (data not shown). The

fact that cells at low density enlarged and did not grow

is consistent with our prior findings on the effect of this

dose of H#O#on these cells at low density (Giblin et al.,

1985). Treatment of the BSO}H#O#

or CDNB}H#O#

cells with any of the antioxidants not only prevented

cell death but permitted a 20-fold increase in cell

number (Fig. 6, far right bar). The protective effect of

nPG on CDNB}H#O#

treated cells is shown in Fig. 6

and is typical for any of the three antioxidants.

The morphological protection afforded by the anti-

oxidants at early times was investigated using time-

lapse video microscopy (data not shown). In these

experiments, 50,000 cells were treated with BSO}H#O#

or with BSO}nPG}H#O#. Cells were examined during

the initial 3 hr following exposure to H#O#. Cells

treated with BSO}H#O#

rounded up and exhibited

clumping of nuclear material. Cells treated with

BSO}nPG}H#O#

were protected and had a normal

morphology. Thus, GSH depletion per se did not result

in cell death, marked growth inhibition or mor-

phological damage in H#O#-treated cells if the cells

were treated with nPG, TEMPOL or deferoxamine.

124 J. R. REDDAN ET AL.

4. Discussion

Lowering the intracellular level of glutathione by two

independent means, either by BSO or CDNB, increased

the susceptibility of cultured lens epithelial cells to

H#O#-induced damage. Cells treated with H

#O#

alone

appeared normal. The mere reduction of GSH by CDNB

or BSO alone was not cytotoxic and did not inhibit

subsequent cell division. This contrasts with work on

NIH 3T3 cells wherein BSO induces an inhibition of

DNA synthesis (Poot et al., 1995). When lens epithelial

cells with lowered glutathione were exposed to H#O#,

the cells exhibited decreased cell-to-cell contact,

nuclear shrinkage and arborization at the phase-

contrast level and extensive nuclear and cytoplasmic

damage when viewed with the transmission electron

microscope. Mitosis was curtailed in cells treated with

BSO}H#O#

or CDNB}H#O#.

The main finding in the present study is that

treatment of the cells containing lowered glutathione

with TEMPOL, deferoxamine or nPG prevented the

morphological damage and cell death induced by H#O#

and permitted proliferation. The morphological dam-

age elicited by BSO}H#O#and the protection from cell

death afforded by all three antioxidants was verified

using time lapse video microscopy and Trypan blue

exclusion. Recent studies suggest that dietary sup-

plementation with antioxidants lowers the risk or

slows the progression of human cataract formation

(Chylack et al., 1998; Taylor et al., 1998; Leske et al.,

1998). Moreover, TEMPOL has been shown to protect

against DNA strand breaks and cataract formation in

the X-rayed rabbit (Sasaki et al., 1998).

Proposed mechanisms by which TEMPOL, defer-

oxamine or nPG protect from oxidative insult have

been put forth (Mitchell et al., 1990; Reddan et al.,

1992, 1993). TEMPOL and deferoxamine prevent the

generation of reactive oxygen species by different me-

chanisms (Reddan et al., 1993). Neither TEMPOL nor

nPG has catalase activity (Reddan et al., 1993), nor do

they interact with H#O#

(Reddan et al., 1993). Both

TEMPOL (Mitchell et al., 1990; Reddan et al., 1993)

and nPG (Reddan et al., 1991, 1998) are SOD mimics.

The superoxide anion which is dismutated by both

TEMPOL and nPG acts to recycle iron from Fe+$ to

Fe+#. By removing the superoxide anion in a cyclic

reaction (Reddan et al., 1992), TEMPOL prevents the

superoxide-mediated reduction of Fe+$ to Fe+#. Defer-

oxamine chelates iron, blocks the reduction of Fe+$ to

Fe+# and has a high specific binding constant for iron

(10$"), whereas TEMPOL has a very low specific

binding constant for the metal (Reddan et al., 1993).

Thus all three compounds investigated in the current

study can prevent the generation of the damaging

hydroxyl radical. Specific details on the mechanism of

nPG will be described separately.

The present results indicate that glutathione de-

pletion with either BSO or CDNB did not affect the

ability of the rabbit lens epithelial cells to remove H#O#

from the culture medium. This was an unexpected

finding since it was apparent that cells with low GSH

levels were significantly more susceptible to H#O#-

induced damage. However, the results parallel those of

others using GSH-depleted mesothelial cells and

fibroblasts (Kinnula et al., 1992; Spitz, Kinter and

Roberts, 1995). Apparently, catalase activity and the

15–20% GSH remaining in the BSO}CDNB-treated

cells were sufficient, in conjunction with glutathione

peroxidase, to metabolize H#O#

at a normal rate. In

contrast, we and others have shown previously that

cultured cells with lowered catalase activity are

significantly inhibited in their ability to remove

extracellular H#O#

(Giblin et al., 1990; Spitz et al.,

1992). The results of the present study, as previously

suggested by Spitz, Kinter and Roberts (1995),

demonstrate that GSH can protect against oxidative

damage independent of its function of H#O#decompo-

sition via glutathione peroxidase. It appears likely that

a lowered level of cellular GSH, while not affecting the

direct removal of H#O#, may inhibit the effective

detoxification of other potentially damaging molecules

such as free radicals or lipid hydroperoxides formed as

a result of H#O#

exposure. Rowley and Halliwell

(1982) have shown that GSH, but only at elevated

concentrations, is a very effective scavenger of the

hydroxyl radical. High levels of glutathione may also

be essential for protecting against oxidant-induced

release of damaging free iron as shown by studies

of GSH-depleted mouse erythrocytes (Ferrali et al.,

1997). Whatever this important additional function of

glutathione may be, it is apparent that TEMPOL, nPG

and deferoxamine can substitute for it in GSH-depleted

cells.

The morphological damage noted in BSO}H#O#

treated cells is similar to results on BCNU}H#O#treated

lens epithelial cells (Giblin et al., 1990). BCNU inhibits

glutathione reductase without influencing the activity

of other antioxidant enzymes (Giblin et al., 1990). The

glutathione level is reduced by 90% when cultured

rabbit lens epithelial cells are exposed to BCNU and

then to a maintained low level of H#O#

(Giblin et al.,

1990). Lens epithelial cells treated with BCNU}H#O#,

exhibit a nearly 85% reduction of reduced glutathione,

a decrease in cell-to-cell contact, membrane blebbing,

swollen mitochondria and a disorganization of the

cytoskeleton (Ikebe et al., 1989; Giblin et al., 1990).

The common feature in the BSO}H#O#

or CDNB}H

#O#

treated cells and BCNU}H#O#

treated cells is a

reduction in intracellular glutathione.

The damage noted here in BSO}H#O#

treated lens

epithelial cells is comparable to the findings of others

using different models. Other cell types that have been

reported to show increased susceptibility to oxidative

damage when glutathione is reduced include cardiac

myocytes (Le et al., 1993), carotid endothelial cells

(Chen et al., 1992), hippocampal cells (Pellmar, Roney

and Lepinski, 1992), and lung, kidney, liver and

pancreas cells (Martensson et al., 1991). The oxidative

PROTECTION IN GSH DEPLETED CELLS 125

stress induced by BSO is protected by GSH ester and

ascorbate (Martensson et al., 1991). Depletion of

glutathione impairs the viability of cancer cells (Arrick

et al., 1982; Revez, Edgren and Wainson, 1994).

Hydroperoxide damage in retinal pigment epithelial

cells can be prevented by administration of GSH or of

the amino acid precursor of glutathione (Sternberg et

al., 1993).

Depletion of GSH accompanies most forms of human

cataract and is associated with cataract formation in

newborn mice treated with BSO (Calvin, Medvedovsky

and Worgul, 1986). The developing lens shows a

more drastic drop in GSH than any other organ

following BSO treatment (Martensson et al., 1989).

Early lens epithelial lesions precede damage to the lens

fibers in BSO treated mouse pups (Laver et al., 1993).

BSO treated mouse pups show decreased protein

synthesis in the lens fibers (Calvin, Viswanathan and

Fu, 1996) and increased proteolysis of crystallins

(Calvin et al., 1992a).

BSO-induced cataracts in rat or mouse pups gene-

rated either in vivo or in vitro appear to be due to

oxidative stress. This interpretation is buttressed by

the finding that the cataracts are prevented by the

administration of glutathione monoester (Martensson

et al., 1989), alpha lipoic acid (Maitra et al., 1995)

and by thiols or ascorbate (Martensson and Meister,

1991; Calvin et al., 1997b). Calvin et al. (1997a),

reported that inhibition of serine-threonine protein

kinase with H7 prevents BSO-induced cataracts in

mouse lenses. H7 is known to block the early response

genes c-jun and c-fos which are associated with a

signaling pathway that may lead to cytotoxicity (Li et

al., 1994).

It is of interest that oxidative stress in cultured

monkey lenses induced by H#O#

or ultraviolet light

increases the levels of Alzheimer’s precursor protein

and beta amyloid (Frederikse et al., 1996). Treatment

of cultured rabbit lens epithelial cells (line N}N1003A,

Reddan et al., 1986) with beta amyloid is cytotoxic

(Frederikse et al., 1996). It has been suggested that the

increase in beta amyloid precursor protein and beta

amyloid may mediate the mechanism by which

oxidative damage leads to cataract formation.

The view is emerging that thiols play a major role in

modulating receptor activity, hormone action, redox

reactions, cell signaling, gene expression, and other

fundamental biological processes including differ-

entiation and cell proliferation (Packer and Cadenas,

1995). In the lens, as in other tissues, glutathione is

multifunctional in that it can detoxify xenobiotics, act

as an antioxidant, and decrease the reaction of lens

proteins with sugars (Harding, Blakytny and Ganea,

1996). Whether the epithelium, which has the highest

concentration of glutathione in the lens (Reddy,

1971), contributes to the maintenance of reduced

glutathione levels in the deeper portions of the organ

remains to be determined. It is possible that decreased

movement of glutathione from the epithelium to the

interior of the lens may be a common feature that

predisposes the organ to cataract formation.

Acknowledgements

We wish to thank Mr Bhargavan Chakrapani and MrTodd Miller for their expert technical assistance. The studywas supported by NIH grants EY00362, EY02027, andEY05230 (CORE grant for vision research). John Pena is aHoward Hughes Undergraduate Fellow.

References

Arrik, B. A., Nathan, C. F., Griffith, O. W. and Cohn, Z. A.(1982). Glutathione depletion sensitizes tumor cells tooxidative cytolysis. J. Biol. Chem. 257, 1231–7.

Calvin, H. I., Medvedovsky, C. and Worgul, B. V. (1986).Near-total glutathione depletion and age-specific cata-racts induced by buthionine sulfoximine in mice. Science233, 553–5.

Calvin, H. I., Viswanathan, K. and Fu, S. C. J. (1996).Modifications in lens protein biosynthesis signal theinitiation of cataracts induced by buthionine sulf-oximine in mice. Exp. Eye Res. 63, 527–38.

Calvin, H. I., Medvedovsky, C., David, J. C., Broglio, T. M.,Hess, J. L., Fu, S. C. J. and Worgul, B. V. (1991). Rapiddeterioration of lens fibers in GSH-depleted mouse pups.Invest. Ophthalmol. Vis. Sci. 32, 1916–24.

Calvin, H. I., Patel, S. A., Zhang, J. P., Li, M. Y. and Fu,S. C. J. (1992a). Progressive modifications of mouse lenscrystallins in cataracts induced by buthionine sulfoxi-mine. Exp. Eye Res. 54, 611–19.

Calvin, H. I., Von Hagen, S., Hess, J. L., Patel, S. A. and Fu,S. C. J. (1992b). Lens GSH depletion and electrolytechanges preceding cataracts induced by buthioninesulfoximine in suckling mice. Exp. Eye Res. 54, 621–6.

Calvin, H. I., Banerjee, U. and Fu, S. C. J. (1997a). Inhibitionof mouse BSO cataracts by the protein-kinase inhibitor,H-7. Invest Ophthalmol. Vis. Sci. 38, 5376.

Calvin, H. I., Zhu, G. P., Wu, J. X., Banerjee, U. and Joseph,S. C. (1997b). Progression of mouse buthionine sul-foximine cataracts in vitro is inhibited by thiols orascorbate. Exp. Eye Res. 65, 341–7.

Chakrapani, B., Yedavally, S., Leverenz, V. R., Giblin, F. J.and Reddy, V. N. (1990). Reduced and oxidized gluta-thione levels in human aqueous and cataracts asdetermined by electrochemical detection. Invest. Oph-thalmol. Vis. Sci. 31, 205.

Chen, S. H., Wu, H. L., Lin, M. T., Jen, C. J., Wing, L. Y. C.,Lei, H. Y., Tsao, C. J. and Chang, W. C. (1992).Cytoprotective effect of reduced glutathione in hydrogenperoxide-induced endothelial cell injury. Prost. Leuk.Essen. Fatty Acids 45, 299–305.

Chylack, L. T., Jr., Schalch, W., Kopcke, W., Phelps-Brown,N., Hurst, M., Mitchell, S., Thien, U., and Bron, A. J., theREACT Group (1998). Roche European-American cat-aract trial (REACT) : Efficacy of an oral antioxidantmicronutrient mixture to slow progression of age-related cataract (ARC). Invest. Ophthalmol. Vis. Sci. 39,S304.

Delamere, N. A., Dean, W. L., Stidam, J. M. and Moseley,A. E. (1996). Influence of amphotericin B on the sodiumpump of porcine lens epithelium. Am. J. Physiol. 270,c465–73.

Ferrali, M., Signorini, C., Caciotti, B., Sugherini, L., Ciccoli,L., Giachetti, D. and Comorti, M. (1997). Protectionagainst oxidative damage of erythrocyte membrane bythe flavonoid quercetin and its relation to iron chelatingactivity. FEBS Lett. 416, 123–9.

126 J. R. REDDAN ET AL.

Frederikse, P. H., Garland, D., Zigler, J. S. and Piatigorsky, J.(1996). Oxidative stress increases production of betaamyloid precursor protein and beta-amyloid (Abeta) inmammalian lenses, and Abeta has toxic effects on lensepithelial cells. J. Biol. Chem. 271, 10169–74.

Giblin, F. J., McCready, J. P., Reddan, J. R., Dziedzic, D. C.and Reddy, V. N. (1985). Detoxification of hydrogenperoxide by cultured rabbit lens epithelial cells : Par-ticipation of the glutathione redox cycle. Exp. Eye Res.40, 827–40.

Giblin, F. J., Reddan, J. R., Schrimscher, L., Dziedzic, D. C.and Reddy, V. N. (1990). The relative roles of theglutathione redox cycle and catalase in the detoxifi-cation of H

#O#

by cultured rabbit lens epithelial cells.Exp. Eye Res. 50, 795–804.

Griffith, O. (1980). Determination of glutathione andglutathione disulfide using glutathione reductase and2-vinylpyridine. Anal. Biochem. 106, 207–12.

Griffith, O. W. (1982). Mechanism of action, metabolism,and toxicity of buthionine sulfoximine and its higherhomologs, potent inhibitors of glutathione synthesis. J.Biol. Chem. 257, 13704–12.

Harding, J. J., Blakytny, R. and Ganea, E. (1996). Gluta-thione in disease. Biochem. Soc. Trans. 24, 881–4.

Horwitz, J., Dovrat, A., Straatsma, B. R., Revilla, P. J. andLightfoot, D. O. (1987). Glutathione reductase in hu-man lens epithelium: FAD-induced in vitro activation.Curr. Eye Res. 6, 1249–56.

Ikebe, H., Susan, S. R., Giblin, F. J., Reddan, J. R. and Reddy,V. N. (1989). Effect of inhibition of the glutathioneredox cycle on the ultrastructure of peroxide-treatedrabbit epithelial cells. Exp. Eye Res. 48, 421–32.

Kinnula, V. L., Everitt, J. L., Mangum, J. B., Chang, L. Y. andCrapo, J. D. (1992). Antioxidant defense mechanisms incultured pleural mesothelial cells. Am. J. Respir. Cell.Mol. Biol. 7, 95–103.

Krausz, E., Augusteyn, R. C., Quinlan, R. A., Reddan, J. R.,Russell, P., Sax, C. M. and Graw, J. (1996). Expressionof crystallins, PAX6, filensin, CP49, MIP and MP20 inlens-derived cell lines. Invest. Ophthalmol. Vis. Sci. 37,2120–8.

Laver, N. M., Robison, W. G., Calvin, H. I., and Fu, S. C. J.(1993). Early epithelial lesions in cataracts of GSH-deleted mouse pups. Exp. Eye Res. 57, 493–8.

Le, C. T., Hollaar, L., van der Valk, E. J. M. and van derLaarse, A. (1993). Buthionine sulfoximine reduces theprotective capacity of myocytes to withstand peroxide-derived free radical attack. J. Mol. Cell. Cardiol. 25,519–28.

Leske, M. C., Chylack, L. T., Jr., He, W., Wu, S. Y.,Schoenfeld, E., Friend, J. and Wolfe, J. (1998). Anti-oxidant vitamins and nuclear opacities : the longitudinalstudy of cataract. Ophthalmology 105, 831–6.

Li, W. C., Wang, G. M., Wang, R. R. and Spector, A. (1994).The redox active components H

#O#

and N-acetyl--cysteine regulate expression of c-jun and c-fos in lenssystems. Exp. Eye Res. 59, 179–90.

Maitra, I., Serbinova, E., Trischler, H. and Packer, L. (1995).alpha-Lipoic acid prevents buthionine sulfoximine-induced cataract formation in newborn rats. FreeRadical Biol. Med. 18, 823–9.

Martensson, J. and Meister, A. (1991). Glutathione defi-ciency decreases tissue ascorbate levels in newbornrats : ascorbate spares glutathione and protects. Proc.Nat. Acad. Sci. USA 88, 4656–60.

Martensson, J., Steinherz, R., Jain, A. and Meister, A. (1989).Glutathione ester prevents buthionine sulfoximine-induced cataracts and lens epithelial cell damage. Proc.Nat. Acad. Sci. USA 86, 8727–31.

Martensson, J., Jain, A., Stole, E., Frayer, W., Auld, P. A. andMeister, A. (1991). Inhibition of glutathione synthesis

in the newborn rat : a model for endogenously producedoxidative stress. Proc. Nat. Acad. Sci. USA 88, 9360–6.

Meakin, S. O., Reddan, J. R., Tsui, L. C. and Breitman, M. L.(1989). A rabbit lens epithelial cell line supportsexpression of an exogenous crystallin gene charac-teristic of lens fiber cell differentiation. Exp. Eye Res. 48,131–7.

Mitchell, J. B., Samuni, A., Krishna, M. C., DeGraff, W. G.,Ahn, M. S., Samuni, U. and Russo, A. (1990). Biologi-cally active metal-independent superoxide dismutasemimics. Biochemistry 29, 2802–7.

Nagaraj, R. H., Sell, D. R., Prabhakaram, M., Ortwerth, B. J.and Monnier, V. M. (1991). High correlation betweenpentosidine protein crosslinks and pigmentation impli-cates ascorbate oxidation in human lens senescenceand cataractogenesis. Proc. Nat. Acad. Sci. USA 88,10257–61.

Packer, L. and Cadenas, E. (1995). Biothiols in health anddisease. Marcel Dekker, Inc : New York, U.S.A.

Pellmar, T. C., Roney, D. and Lepinski, D. L. (1992). Role ofglutathione in repair of free radical damage in hip-pocampus in vitro. Brain Res. 583, 194–200.

Poot, M., Teubert, H., Rabinovitch, P. S., and Kavanagh,T. J. (1995). De novo synthesis of glutathione is requiredfor both entry into and progression through the cellcycle. J. Cell. Physiol. 163, 555–60.

Rathbun, W. B. and Murray, D. L. (1991). Age-relatedcysteine uptake as rate-limiting in glutathione synthesisand glutathione half-life in the cultured human lens.Exp. Eye Res. 53, 205–12.

Rathbun, W. B., Schmidt, A. J. and Holleschau, A. M.(1993). Activity loss of glutathione synthesis enzymesassociated with human subcapsular cataract. Invest.Ophthalmol. Vis. Sci. 34, 2049–54.

Reddan, J. R. (1982). Control of cell division in the ocularlens, retina and vitreous. In: Cell biology of the eye.(McDevitt, D. Ed.). Pp. 299–375. Academic Press : NewYork, USA.

Reddan, J. R., Chepelinsky, A. B., Dziedzic, D. C., Piatigorsky,J. and Goldenberg, E. M. (1986). Retention of lensspecificity in long-term cultures of diploid rabbit lensepithelial cells. Differentiation 33, 168–74.

Reddan, J. R., Giblin, F. J., Dziedzic, D. C., McCready, J. P.,Schrimscher, L. and Reddy, V. N. (1988). Influence ofthe activity of glutathione reductase on the response ofcultured lens epithelial cells from young and old rabbitsto hydrogen peroxide. Exp. Eye Res. 45, 209–21.

Reddan, J. R., Sevilla, M. D., Giblin, F. J., Padgaonkar, V.,Dziedzic, D. C., Leverenz, V. R., Misra, I. C. and Peters,J. L. (1991). SOD mimics protect lens epithelial cells fromH

#O#

insult. Invest. Ophthalmol. Vis. Sci. 32, 849.Reddan, J., Sevilla, M., Giblin, F., Padgaonkar, V., Dziedzic,

D. and Leverenz, V. (1992). TEMPOL and deferoxamineprotect cultured rabbit lens epithelial cells from H

#O#

insult : Insight into the mechanism of H#O#-induced

injury. Lens Eye Tox. Res. 9, 385–93.Reddan, J. R., Sevilla, M. D., Giblin, F. J., Padgaonkar, V.,

Dziedzic, D. C., Leverenz, V., Misra, I. C. and Peters, J. L.(1993). The superoxide dismutase mimic TEMPOLprotects cultured rabbit lens epithelial cells fromhydrogen peroxide insult. Exp. Eye Res. 56, 543–54.

Reddan, J. R., Giblin, F. J., Sevilla, M. D., Pena, J. T.,Leverenz, V. R. and Dziedzic, D. C. (1998). n-Propylgallate curtails oxidative damage in cultured lensepithelial cells. Molec. Biol. Cell 9, 499a.

Reddy, V. N. (1971). Metabolism of glutathione in the lens.Exp. Eye Res. 11, 310–28.

Reddy, V. N., Garadi, R., Chakrapani, B. and Giblin, F. J.(1988). Effect of glutathione depletion on cationtransport and metabolism in the rabbit lens. Ophthal.Res. 20, 191–9.

PROTECTION IN GSH DEPLETED CELLS 127

Revez, L., Edgren, M. R. and Wainson, A. A. (1994).Selective toxicity of buthionine sulfoximine (BSO) tomelanoma cells in vitro and in vivo. Int. J. RadiationOncol. Biol. Phys. 29, 403–6.

Rowley, D. A. and Halliwell, B. (1982). Superoxide-dependent formation of hydroxyl radicals in the presenceof thiol compounds. FEBS Lett. 138, 33–6.

Sasaki, H., Lin, L. R., Yokoyama, T., Sevilla, M. D., Reddy,V. N. and Giblin, F. J. (1998). TEMPOL protects againstlens DNA strand breaks and cataract in the x-rayedrabbit. Invest. Ophthalmol. Vis. Sci. 39, 544–52.

Sharma, Y., Druger, R., Mataic, D., Bassnett, S. and Beebe,D. C. (1997). Aqueous-humor hydrogen-peroxide andcataract. Invest. Ophthalmol. Vis. Sci. 38, 5372.

Spector, A. (1991). The lens and oxidative stress. In:Oxidative stress : Oxidants and antioxidants. (Sies, H. Ed.).Pp. 529–58. Academic Press, London, USA.

Spector, A., Ma, W. C. and Wang, R. R. (1998). The aqueoushumor is capable of generating and degrading H

#O#.

Invest. Ophthalmol. Vis. Sci. 39 : 1188–97.Spitz, D. R., Adams, D. T., Sherman, C. M. and Roberts, R. J.

(1992). Mechanisms of cellular resistance to hydrogen

peroxide, hyperoxia, and 4-hydroxy-2-nonenal toxicity :the significance of increased catalase activity in H

#O#-

resistant fibroblasts. Arch. Biochem. Biophys. 292,221–7.

Spitz, D. R., Kinter, M. T. and Roberts, R. J. (1995).Contribution of increased glutathione content tomechanisms of oxidative stress resistance in hydrogen-peroxide resistant hamster fibroblasts. J. Cell. Physiol.165, 600–9.

Sternberg, P., Davidson, P. C., Jones, D. P., Hagen, T. M.,Reed, R. L. and Drews-Botsch, C. (1993). Protection ofretinal pigment epithelium from oxidative injury byglutathione and precursors. Invest. Ophthalmol. Vis. Sci.34, 3661–8.

Taylor, A., Jacques, P., Chylack, L. T., Wolfe, J., Friend, J.,Raghavan, A., Balaram, M., Willett, W. and Hankison,S. (1998). Use of vitamin C supplements for "10 yearsis associated with diminished risk for early age-relatedcataract. Invest. Ophthalmol. Vis. Sci. 39, s241.

Tietze, F. (1969). Enzymic method for quantitative de-termination of nanogram amounts of total and oxidizedglutathione. Anal. Biochem. 27, 502–22.