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ICANCÕR
RESEARCH
(SUPPL.)
54,
Glutathione, Ascorbate, and Cellular Protection1
Alton Meister
Department of Biochemistry,
Cornell University Medical College, New York, New York 10021
Introduction
Glutathione. a tripeptide thiol present in virtually all animal cells, is
synthesized within many cells from its constituent amino acids (glu
tamate, cystcine, glycine); these "nonessential" amino acids can be
synthesized within the body and are also obtained from the diet.
Glutathione is also synthesized by tumors, some of which (notably
drug- and radiation-resistant tumors) exhibit high cellular levels of
glutathione and high capacity for the synthesis of glutathione. We
reported at previous conferences in this series on the increase of
cellular radiosensitivity that occurs after administration of an inhibitor
of glutathione synthesis (1, 2) and on the effects of modulation of
glutathione metabolism (3, 4). These and related topics (5, 6) will be
summarized here with emphasis on some current developments.
Glutathione is probably the most important cellular antioxidant.
Interestingly, Fahey and Sundquist (7) found strong evidence for an
evolutionary link between glutathione and aerobic eukaryotic metab
olism; the findings indicate that glutathione evolved as a molecule that
protects cells against oxygen toxicity. Although there is currently
much interest in the hypothesis that oxidative phenomena may lead to
a variety of pathological states, and that antioxidants may play a
significant protective role, the important role that glutathione plays in
the protection of cells has sometimes been insufficiently appreciated.
Cells that are deprived of glutathione typically suffer severe oxidative
damage associated with mitochondria! degeneration. Analogous ef
fects are not always found when there is a deficiency of certain other
cellular components that are thought to act as antioxidants. It has long
been known that the antioxidant ascorbate is required in the diet of
humans and certain other animals such as the guinea pig (but not by
many other animals, including some commonly used in laboratory
experiments; e.g., mice, rats, rabbits). The ascorbate deficiency syn
drome, scurvy, which is associated with oxidative inactivation of
certain enzymes, can be prevented in humans by administration of as
little as 10 mg/day of ascorbate. The officially recommended daily
dose of ascorbate for humans is 30-100 mg/day (depending upon the
a-tocopherol in its reduced form, either by a direct reaction or by a
pathway involving ascorbate (10-17). Glutathione. which has the
important function of maintaining the reducing milieu of cells, is
undoubtedly involved in the reduction of many cellular components;
e.g., other tocopherols and ß-carotenearc apparently also maintained
by glutathione-mediated reactions (e.g.. Réf.18).
An interesting aspect of glutathione metabolism and function re
lates to drug-resistant and radiation-resistant tumors that have high
levels of glutathione or exhibit high capacity for glutathione synthesis.
Such tumors have a greater requirement for glutathione than do many
normal tissues and this provides a promising chemotherapeutic ap
proach, which is considered below.
Biochemistry of Glutathione: Enzymology and Transport
Phenomena
Fig. 1 gives some of the biochemical transformations of glutathi
one, which is synthesized in two steps from glutamate, cystcine, and
glycine (Reactions / and 2). Metabolic utilization of glutathione
follows several pathways including reactions catalyzed by the gluta
thione S-transferases (mercapturate pathway). Glutathione is a sub
strate of the glutathione peroxidases which destroy hydrogen peroxide
and organic peroxides. The glutathione disulfide formed is reduced to
glutathione in an NADPH-mediated reaction (Fig. 2). Glutathione not
only provides reducing power needed for the conversion of dehy
droascorbate to ascorbate, but also for the conversion of ribonucleotides to deoxyribonucleotides and for a variety of thiol-disulfide
interconversions; glutathione is therefore important for the synthesis
and repair of DNA, and for the folding of newly synthesized proteins.
The utilization of glutathione (Fig. 1; y-glutamyl cycle) is initiated
extracellularly by the actions of y-glutamyl transpeptidase and dipeptidase; these enzymes are bound to the outside of cell membranes. The
transpeptidase acts on glutathione. glutathione disulfide and glutathi
one 5-conjugates. The reactions catalyzed by y-glutamyl transpepti
dase take place in the presence of amino acids and lead to the
formation of y-glutamyl amino acids (19). Cystine is the most active
country); although much larger doses of ascorbate than this are taken
amino acid acceptor of the y-glutamyl group (20); other neutral amino
by some individuals, it is estimated that a substantial proportion of the
acids such as methionine and glutamine are also good acceptors (21).
human population takes in relatively small amounts. The question as
y-Glutamyl amino acids are transported and become substrates of
to whether larger doses of ascorbate and also of other "antioxidants"
y-glutamyl cyclotransferase, which converts y-glutamyl amino acids
would have beneficial effects has often been discussed but remains
into 5-oxoproline and the corresponding free amino acids (22-25).
unsettled.
5-Oxoproline is converted to glutamate in the reaction catalyzed by
Experimental findings summarized here that are relevant to this
5-oxoprolinase (26, 27). Of the several reactions of the y-glutamyl
question include: (a) the observation that glutathione deficiency in cycle, three require ATP, which is split to ADP and P¡(Reactions /,
animals that are unable to synthesize ascorbate (newborn rats, guinea
2 and 6).
pigs) is lethal and that death can be prevented by giving high doses of
It is notable that y-glutamyl transpeptidase is mainly extracellularly
ascorbate; and (b) the onset of scurvy in guinea pigs that are fed a diet
located, whereas glutathione is found principally within cells. Many
deficient in ascorbate is substantially delayed by giving glutathione
cells normally export glutathione. An early observation that led to the
monoethyl ester, a glutathione delivery agent (6, 8).
discovery of such transport was the finding of marked glutathionuria
Various questions about the functions of putative antioxidant com
and glutathionemia after administration of inhibitors of y-glutamyl
pounds need to be considered in relation to the functions of cellular
transpeptidase to experimental animals (28-30). Interestingly, the
glutathione. As discussed here, one such function, shown in vivo (9),
urine of animals given such inhibitors contains cysteine and y-gluis to reduce dehydroascorbate to ascorbate. Glutathione also keeps
tamylcysteine moieties as well as glutathione. Patients who are defi
cient in transpeptidase show similar findings (30). The physiological
1 Presented at the 4th International Conference on Anticarcinogenesis & Radiation
function of y-glutamyl transpeptidase is thus closely connected with
Protection. April 1H-23, 1993, Baltimore. MD. The research described here that was
the metabolism and transport of glutathione. When y-glutamyl
carried out in the author's laboratory was supported in part by NIH Grant 2 R37 DK12034
from the United States Public Health Service.
transpeptidase is markedly decreased, there is a substantial loss of
1969s
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GLUTATHIONE,
ASCORBATE,
AND CELLULAR
OXIDATION-REDUCTION
PATHWAYS
PROTECTION
bile. Plasma glutathione is used by many tissues, e.g., kidney, lung,
and brain. Glutathione itself is not significantly transported into most
of the cells of these tissues but is broken down by membrane-bound
y-glutamyl transpeptidase and dipeptidase; the products of breakdown
GSSG
are transported and utilized for glutathione synthesis. This is an
important pathway of glutathione metabolism.
Effects of Glutathione Deficiency
Information relevant to this topic has come from observations on
human mutants that have decreased levels of glutathione (e.g., pa
tients with glutathione disulfide reducÃ-asedeficiency, glucose-6-phosphate dehydrogenase deficiency, deficiencies of y-glutamylcysteine
synthetase and of glutathione synthetase) (34), and from experimental
studies on animals in which glutathione deficiency was produced (5).
Humans deficient in glutathione may exhibit increased tendency to
hemolysis, cataracts, and central nervous system abnormalities. In one
condition (glutathione synthetase deficiency) there is a secondary
metabolic acidosis, often life-threatening, due to overproduction of
5-oxoproline. Glutathione levels are markedly reduced in some of
2HJ
MERCAPTURATE
PATHWAY
à )/
Glutamate
Y-Glu-AA
these patients, but in none of them is glutathione completely absent.
Experimental production of glutathione deficiency has been at
tempted by administration of compounds that react with glutathione
(e.g., diethyl maléate,phorone) and oxidizing agents (e.g., diamide
and t-butyl hydroperoxide). However, as discussed elsewhere (5),
such nonspecific agents have major limitations. Thus far, the most
useful approach to production of glutathione deficiency has been
treatment with specific inhibitors of y-glutamylcysteine synthetase,
the enzyme that catalyzes the first and rate-limiting step of glutathione
synthesis.
The discovery that methionine sulfoximine inhibits y-glutamylcys
teine synthetase followed from previous studies which showed that
this enzyme, like glutamine synthetase, involves a mechanism in
which y-glutamyl phosphate is an intermediate (35). Studies with
various substrates and substrate analogues led to mapping of the
Fig. 1. Overall pathway of glutathione (GSH) metabolism. /. y-glutamylcysteine
synthetase; 2, glulathione synthetase; 3, y-glutamyl transpeptidase; 4, cysteinyl glycine
hydrolases; 5, y-glutamyl cyclotransfera.se; 6, 5-oxoprolinase; 7, glutathione 5-transferases; 8, transport and reduction of y-Glu-(Cys)2; 9, see Fig. 2. Reactions 1, 2, and 6
involve cleavage of ATP to ADP and P,.
-GSSG
NADPH, H
Deoxyribonucleottd
NADP
V-GLU CYCLE
Fig. 2. Oxidation reduction pathways. These reactions involve those catalyzed by
glutathione transhydrogenases, glutathione peroxidases (Se-containing and others), and
glutathione disulfidc reducÃ-ase.
cysteine moieties. The pathway (illustrated in Fig. 3), which was first
elucidated in studies on the kidney, serves as a recovery system for
cysteine moieties (31). Thus, exported glutathione interacts with cystine and y-glutamyl transpeptidase to produce y-glutamylcystine
which is transported. High levels of extracellular glutathione inhibit
such transport (32). y-Glutamylcystine is reduced intracellularly to
yield cysteine and y-glutamylcysteine, both of which are used for the
synthesis of glutathione. The cysteinylglycine formed in the transpeptidation reaction is split extracellularly to form cysteine and glycine;
this reaction may also occur intracellularly after transport of the
dipeptide. y-Glutamyl transpeptidase and cysteinylglycine dipeptidase activity thus function in the recovery of cysteine moieties that are
necessary for the functioning of a recycling pathway involving syn
thesis and export of glutathione (33).
Glutathione is exported by the liver to the blood plasma and to the
CyS CyS -
CySH-Gly
Fig. 3. "Salvage" pathway. Glutathione (GSH), synthesized intracellularly (reactions /
and 2), is exported (3) and reacts with y-glutamyl transpeptidase and extracellular cystine
(4) to produce y-glutamylcystine, which is transported (5) and reduced intracellularly (7)
to form cysteine and y-glutamylcysteine. Transport of y-glutamylcystine is inhibited by
extracellular GSH (6). Experiments in which y-glutamylcystine was selectively labeled
with 15S on the internal (Sf) or external (S*) S atom gave results consistent with this
pathway (31). A block at step 4 (reaction catalyzed by y-glutamyl transpeptidase) leads to
accumulation of glutathione and to urinary excretion of glutathione and cysteine and
y-glutamylcysteine moieties (see text).
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OLUTATHIONE,
ASCORBATE.
active site ofglutamine synthetase and to design of selective inhibitors
of glutamine synthetase (a-ethyl methionine sulfoximine) and of
y-glutamylcysteine synthetase [prothionine sulfoximine, BSO,2 and
others (35-40)]. BSO and related compounds have very little or
virtually no effect on glutamine synthetase but inhibit y-glutamylcys
teine synthetase very effectively. BSO, now commercially available,
has been widely used in in vitro experiments and in vivo. The effects
of glutathione deficiency induced by administration of BSO have been
extensively examined (see Ref. 5) and include the following:
1. Glutathione deficiency sensitizes cells to the effects of radiation,
oxidative reactions, and to various toxic compounds. These effects
have been applied in chemotherapy and radiation systems (5, 41-44).
Tumor cell resistance associated with overproduction of glutathione is
reversed by treatment with BSO in animal models and is currently
being tested clinically (45). Depletion of glutathione by treatment with
buthionine sulfoximine sensitizes cells to the toxic effects of heavy
metals (46, 47), nitrogen mustards (48, 49), radiation (1, 2, 5),
cisplatin (50), cyclophosphamide (51, 52), morphine (53), compounds
that produce oxidative cytolysis (54), and others (55).
2. Glutathione deficiency leads to oxidative stress in many tissues
(5). Mitochondria! and associated cell damage is found in mice treated
with BSO. Mitochondria do not synthesize glutathione but obtain it by
transport from the cytosol. Several tissues of adult mice are affected
by administration of BSO, but in newborn rats and guinea pigs more
extensive damage is found and there is early mortality due to multiorgan failure (56). Glutathione deficiency in these experimental sys
tems serves as a model of endogenously produced oxidative stress.
Mortality and tissue damage are significantly decreased by adminis
tration of glutathione esters or of ascorbate (5, 9).
3. Deficiency of glutathione leads to decreased reduction of dehydroascorbate to ascorbate in vivo. This is observed in newborn rats and
guinea pigs, animals that cannot synthesize ascorbate, and also in
adult mice, which can (6). In adult mice, glutathione deficiency leads
to induction of ascorbate synthesis in the liver (57), and this explains
why BSO has less damaging effects on adult mice than it does on
newborn rats and guinea pigs.
4. Glutathione deficiency in newborn rats and mice leads to for
mation of ocular cataracts (9, 58, 59). Cataracts have also been found
in some patients with inherited glutathione disulfidc reducÃ-asedefi
ciency.
5. Treatment of peripheral blood mononuclear cells with BSO was
found to markedly inhibit their proliferation (4), and later work has
confirmed that glutathione deficiency decreases lectin-induced prolif
eration of lymphocytes (60). Although glutathione is required for
proliferation, the mechanism of its function in this system is still
unsettled.
AND CELLULAR
PROTECTION
these agents might make tumor cells more sensitive to chemotherapy
and to radiation treatment (41 ). The potential usefulness of BSO in the
sensitization of cells to radiation was first directly shown in studies on
several human lymphoid cell lines (1, 2). Cells that had 4-5% of the
control levels of glutathione were found to be much more sensitive
than the controls to the effects of y-irradiation. It was found that
treatment with BSO of mice bearing B16 melanomas sensitizes this
radio-resistant tumor to radiation (61, 62). Treatment with BSO alone
did not affect tumor growth, but treatment with BSO and radiation led
to significant decrease in the size of the tumor and to an increase in the
longevity of the tumor-bearing mice. It was also found that treatment
of resistant Icukemias with BSO led to sensitization of the tumors to
phenylalanine mustard (48. 49). In studies with mice bearing such
resistant tumors, i.p. infusion of BSO led to sensitization of the tumors
and to an increase in the life span of the treated animals. Additional
studies on the relationship between glutathione levels and the expres
sion of primary drug resistance and cross-resistance in human ovarian
cancer cell lines, together with studies in which an i.p. model of
human ovarian cancer was developed in nude mice, were of impor
tance because they led to a clinical trial of BSO which is now in
progress (44, 45). In these studies it was also found that resistance of
the tumors to phenylalanine mustard is associated with resistance to
other drugs such as Adriamycin. Of interest, these drug-resistant cells
are also resistant to radiation. It seems probable that at least one type
of multidrug resistance is associated with overproduction of glutathi
one; however, other cellular mechanisms can also lead to drug resis
tance. Much attention has been given to multidrug resistance associ
ated with a novel membrane glycoprotein; this type of resistance is
associated with decreased accumulation within the tumor of a number
of structurally unrelated drugs. However, this P-glycoprotein-related
system does not appear to confer radiation resistance.
Cellular levels of glutathione may determine the degree of drug
resistance or radiation resistance of a particular tumor, but the capac
ity of a tumor cell to synthesize glutathione may also be an important
factor in resistance. The ability of a cell to synthesize glutathione
rapidly in response to a stress may be as important or perhaps more
important than the initial cellular level of glutathione. This idea is
supported by model studies on a strain of Escherichia coli enriched in
its content of y-glutamylcysteine synthetase and glutathione syn
thetase by recombinant DNA techniques (63). Recent studies on
tumors that are resistant and sensitive to Adriamycin are consistent
with this idea (5, 64).
Studies on human ovarian tumor cell lines that are resistant to
cisplatin showed that cellular glutathione levels are greatly
increased (13- to 50-fold) as compared with the sensitive cells of
origin (65). The cell lines examined exhibited up to 1000-fold
increases in resistance to cisplatin. Cisplatin resistance was
associated
with increased
expression
of mRNAs
for yApplication of Glutathione Depletion to the Treatment of
glutamylcysteine
synthetase
and
y-glutamyl
transpeptidasc
and
with
Tumors: Sensitization of Tumors to Chemotherapy
increased activities of these enzymes. Thus, y-glutamylcysteine
and to Radiation
synthetase and y-glutamyl transpeptidase appear to contribute to the
Early studies showed that tumor cells that are resistant to alkylating
development of cisplatin resistance. It is notable that there was a
agents have increased levels of nonprotein thiol, later shown to be significant increase in the levels of both subunits of yglutathione (see Ref. 5). The resistance of leukemic cells to phenylglutamylcysteine synthetase. The heavy subunit (Mr 72,614) of this
alanine mustard was found not to be related to an effect on uptake or enzyme (66) contains the binding sites for the substrates (ATP,
efflux of the mustard, but to be closely related to the cellular level of glutamate, cysteine) and is feedback inhibited by glutathione (67).
glutathione (48, 49). The resistant cells converted phenylalanine mus
However, the light subunit (Mr 30,548) of this enzyme is required
tard to a nontoxic compound in a glutathione-dependent dehydrochlofor optimal activity and for physiologically appropriate feedback
rination reaction.
inhibition (68). It seems therefore to function in a regulatory
After development of the amino acid sulfoximine inhibitors of
manner.
y-glutamylcysteinc synthetase, it was suggested that treatment with
Because the effects of glutathione deficiency induced in experi
mental animals by administration of BSO can be reversed to a sig
~ The abbreviation used is: BSO. buthionine sulfoximine.
nificant extent by administration of large amounts of ascorbate (sec
1971s
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OLUTATHIONE.
ASCORBATE,
below), it is relevant to consider the ascorbate status of patients treated
with BSO (see Ref. 69). It is possible that administration of large
doses of ascorbate to such patients would negate the desired effects of
BSO administration.
AND CELLULAR
PROTECTION
Table 1 Effects of glutathiurie deficiency
EffectDeath,
treatment with BSO leads to additional and gradual decline of cellular
glutathione. This biphasic decrease in the cellular glutathione level led
to further investigations which showed that a substantial fraction of
cellular glutathione is sequestered in the mitochondria. BSO is not
transported significantly into mitochondria (70). However, mitochon
dria were found to lack the enzymes required for glutathione synthe
sis; therefore, the failure of BSO to enter mitochondria is not relevant,
but the absence of the synthetases from mitochondria showed that
mitochondrial glutathione must arise from the cytosol (71). Studies on
isolated rat liver mitochondria indicate that mitochondrial glutathione
homeostasis is regulated by a multicomponent transport system which
appears to explain the remarkable ability of mitochondria to take up
and to retain glutathione (72). Evidence was found for two transport
ers with apparent Km values of 60 ¡JLM
and 5.4 mM. Extramitochondrial
glutathione promotes mitochondrial uptake and exchange, and the
intermembranous space appears to function as a recovery zone that
facilitates efficient cycling of matrix glutathione. Decreased levels of
glutathione produced by administration of BSO decrease the net
export of glutathione from mitochondria to the cytosol. That the net
efflux of glutathione from mitochondria is very slow when there are
low levels of extramitochondrial glutathione is consistent with a
mechanism that conserves mitochondrial glutathione during periods
of cytosolic glutathione depletion.
A significant fraction of the oxygen utilized by mitochondria (about
2-5%) is converted, apparently through Superoxide, to hydrogen
peroxide (73). When glutathione levels are greatly decreased, hydro
gen peroxide accumulates, and this leads to extensive mitochondrial
damage. Other antioxidants may be involved in the protection of
mitochondria, but glutathione appears to be the principal functional
one. Mitochondria do not contain catalase and are therefore largely, if
not entirely, dependent upon glutathione and glutathione peroxidases.
Electron microscopy has revealed that mitochondrial damage is an
important consequence of glutathione deficiency in many tissues.
These effects, which are produced without application of external
stress, develop after glutathione is depleted by administration of BSO
(5). Not only mitochondria but other types of cellular damage were
found, including nuclear effects, and in the lungs, effects on the
lamellar bodies. There appears to be a relationship between the extent
of mitochondrial depletion of glutathione and cellular damage, as
estimated by determinations of citrate synthetase and electron micros
copy. In studies on adult mice, degeneration of skeletal muscle was
found when the mitochondrial glutathione levels were decreased to
about 20% of the controls. Similarly, mitochondrial and lamellar body
damage in lung type II cells were found when the mitochondrial
glutathione levels were about 21% of the controls. Jejunal mucosal
damage was found with mitochondrial glutathione levels of about
13% of the controls. In newborn rats, cataracts appeared when the lens
mitochondrial glutathione levels were about 20% of the controls.
A summary of the effects of glutathione deficiency induced by
administration of BSO is given in Table 1. In adult mice, prolonged
treatment with BSO did not produce cellular damage in the liver,
heart, or kidneys. In both newborn rats and guinea pigs, treatment with
BSO led to death within 4-6 days, whereas adult mice did not exhibit
Guinea
mice000+M0-+IIII+
'LiverKidneyLungBrainLensSkeletal
damage
++
++
++
+
0+
0II
On the Essentiality of Glutathione: Mitochondria! Function
When BSO is administered to rats or mice there is rapid decline of
the glutathione levels of the liver and kidney to an apparent limiting
value of 15-20% of the total glutathione initially present. Further
pigs-f-a
rats
++
daysCell
4-6
+
muscleJejunum,
colonHeartStomachLymphocytes"
0Ãœas
+, cell damage or
deathMainly
indicated.Adult
mitochondrial.Newborn
early mortality. The lethal effects of glutathione deficiency in animals
such as newborn rats and guinea pigs (which do not synthesize
ascorbate) appear to be related to multiorgan failure involving mito
chondrial and other damage in liver, kidney, and lung (56). In liver
there is focal necrosis: in kidney, there is proximal tubular damage;
and in lung, there is lamellar body degeneration. The lungs of adult
mice showed damage, although less than in newborn rats.
The mitochondrial and other cell damage seen in newborn animals
and in adults were prevented by administration of glutathione esters or
by administration of ascorbate (see below).
Function of Glutathione in the Reduction of Dehydroascorbate
A function of glutathione in the reduction of dehydroascorbate was
suspected by early investigators. Borsook et al. (74) concluded that
glutathione is involved in the reduction of dehydroascorbate by ani
mal tissues, but Guzman-Barron. another early pioneer in this field,
considered this unlikely (75). Hopkins and Morgan (76) studied this
reaction in plants. The reduction of dehydroascorbate to ascorbate was
examined by a number of investigators in in vitro animal systems (see
Ref. 6), and purified preparations of glutaredoxin and protein disulfide
isomerase were found to exhibit substantial glutathione-dependent
dehydroascorbate reducÃ-aseactivity (77).
Convincing evidence linking glutathione to the reduction of dehy
droascorbate in vivo has recently been obtained (6, 9). In the first of
these studies, newborn rats treated with BSO were found to have
marked depletion of tissue (liver, kidney, lung, brain, eye) ascorbate.
Both the levels of ascorbate and total ascorbate (ascorbate plus dehy
droascorbate) were decreased (6). It is of interest that when ascorbate
was also given to these newborn rats, the levels of glutathione in the
tissues and in their mitochondria were increased significantly, indi
cating that ascorbate can spare glutathione. Findings closely similar to
those made on newborn rats were made in adult guinea pigs (78). In
this species also, tissue damage and early mortality due to glutathione
deficiency are greatly decreased or prevented by administration of
ascorbate. Treatment with ascorbate spares mitochondrial glutathione
as found also in newborn rats (9).
Although glutathione deficiency is lethal to newborn rats and
guinea pigs, adult mice are able to survive because they can synthesize
ascorbate. Treatment of adult mice with BSO actually leads to an
initial increase of the ascorbate level in the liver (57). Within 4 h after
BSO administration, the level of ascorbate in the liver increases about
2-fold and then decreases with concomitant accumulation of dehy
droascorbate. In other tissues, the ascorbate levels decreased and the
levels of dehydroascorbate increased. Therefore, an early effect of
glutathione deficiency in adult mice appears to be an induction of
ascorbate synthesis in the liver. Such induction does not occur in
newborn rats or in guinea pigs, findings consistent with the view that
1972s
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OLUTATHIONE, ASCORBATE. AND CELLULAR PROTECTION
GLU+CySH
GLY
>- GSSG
—-GLUTAREDOXIN ""
DEHYDROASCORBATE
ASCORBATE
H202
BREAKDOWN
Fig. 4. Relationships between glutathione and ascorbatc (from Ref. l>).
these animals do not synthesize ascorbate. Thus, the findings summa
rized in Table 1 appear to be closely connected with the ability of the
experimental animals to synthesize ascorbate. However, glutathione
deficiency in adult mice is not fully compensated by increased ascor
bate synthesis. Thus, electron microscopy of the lungs of adult mice
treated with BSO showed substantial damage to type II cell lamellar
bodies (79). There was also decreased formation of intraulvcolar
tubular myelin, which is secreted by the lamellar bodies. Phosphatidylcholine. the main constituent of lungs surfactant, is synthesized
within the lamellar bodies and is secreted as a protein complex into
the alveolar subphase where it is transformed into tubular myelin.
Markedly decreased levels of phosphatidylcholine were found in the
lungs of adult mice and in the bronchoalveolar fluid after treatment
with BSO. Simultaneous treatment with ascorbate prevented the de
crease in phosphatidylcholine levels in the lung as well as cellular
damage (80).
The reactions given in Fig. 4 appear to account for the finding that
ascorbate can spare glutathione. That glutathione can spare ascorbate
was shown in studies on guinea pigs given a diet deficient in ascorbate
(8). These animals typically develop scurvy after 14-20 days. How
ever, when treated with glutathione monoethyl ester, the onset of
scurvy was significantly delayed (to at least 40 days).
Modulation of Glutathione Metabolism
As discussed above, tumors that require high levels of cellular
glutathione or high cellular capacity for glutathione synthesis are
placed at a disadvantage by treatments that decrease cellular synthesis
of glutathione. On the other hand, most normal cells have a large
excess of glutathione. As suggested previously (3), tumors that lack or
have low levels of catalase might be successfully treated with BSO
alone. It is of interest that BSO exhibits a high degree of inhibitory
activity against melanoma-derived cell lines (81-83). In one study,
seven human melanoma cell lines were found to be sensitive to BSO
and it was suggested that BSO may be an effective agent for mela
noma. Although no simple relationship between glutathione metabo
lism and sensitivity to BSO was recognized in human melanoma cells
(81), further studies may reveal such a connection. Overproduction of
melanin in these cells may be accompanied by increased formation of
products that are detoxified by reactions involving glutathione. The
process of melanin formation involves increased utilization of gluta
thione, either as a participant in the synthesis of melanin (e.g., in the
reaction of glutathione or of cysteine derived from it, with dopaquinone), or as a consequence of increased oxidative phenomena asso
ciated with melanin synthesis. Melanin formation may increase the
requirement for glutathione by the melanoma cell; decreasing gluta
thione synthesis by giving BSO would thus be expected to affect the
melanoma cell much more than normal cells, which have a lower
requirement for glutathione.
Cellular glutathione levels may be increased by administration of
compounds that serve as precursors of cysteine, such as A'-acetyl-Lcysteine and L-2-oxothiazolidine-4-carboxylate (5, 42, 84). Utilization
of the latter compound requires the activity of 5-oxoprolinase which
converts i.-2-oxothiazolidine-4-carboxylate
to i.-cysteine. Certain ex
perimental tumors seem to lack or to be markedly deficient in 5-oxo
prolinase activity, whereas most normal tissues convert this com
pound effectively to L-cysteine, which is effectively used for
glutathione synthesis. A combination therapy involving the use of an
antitumor compound plus the oxothiazolidine has been previously
suggested (3, 4); according to this strategy, normal tissues may use
this compound to increase glutathione synthesis, which tumors would
not be able to do, and this would be expected to increase the thera
peutic effectiveness of anticancer agents. Glutathione levels may
also be increased by treatment with y-glutamylcystinc and related
compounds (31). This approach has the advantage of bypassing
the feedback-inhibited step of glutathione synthesis catalyzed by
y-glutamylcysteine synthetase. Glutathione levels may also be in
creased by administration of glutathione esters, which (in contrast to
glutathione) are well transported into many types of cells and split to
form glutathione (85-87).
Although modulation of glutathione metabolism has largely fo
cused on efforts to decrease or to increase cellular glutathione levels,
other modulations have been considered (3, 4). For example, inhibi
tion of y-glutamyl transpeptidase would be expected to interrupt the
"salvage" pathway of glutathione synthesis (Fig. 3). Since certain
tumors have increased levels of y-glutamyl transpeptidase. it might be
thought that this enzyme would be u promising target for chemother
apy. Patients with inborn deficiency of y-glutamyl transpeptidase
excrete large amounts of glutathione, y-glutamylcysteine, and cys
teine moieties in their urine, and analogous effects have been observed
in experimental animals treated with inhibitors of y-glutamyl
transpeptidase (see above). Compounds currently available for inhi
bition of y-glutamyl transpeptidase include a combination of serine
and borate, but rather high concentrations of these are needed for
effective inhibition. Acivicin is a more effective inhibitor, but this
compound is nonspecific and inhibits a number of glutamine amidotransferases. Acivicin has been tested as an anticancer compound
apparently without success. Other compounds that inhibit y-glutamyl
transpeptidase include 6-diazo-5-oxo-i.-norlcucine and azaserine. An
other potentially effective approach is administration of y-glutamyl
amino acids, which compete with the natural substrates and thus
produce glutathionuria (see Ref. 88).
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Glutathione, Ascorbate, and Cellular Protection
Alton Meister
Cancer Res 1994;54:1969s-1975s.
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