Vol. 166, No. 2, 1990 January 30, 1990
GLUTATHIONE
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 960-966
DISULFIDE ENHANCES THE REDUCED GLUTATHIONE INHIBITION PEROXIDATION IN RAT LIVER MICROSOMES
Richard
W. Scholz*,
Kenneth
S. Graham
and C. Channa
Environmental Resources Research Institute Department of Veterinary Science The Pennsylvania State University University Park, Pennsylvania 16802 Received
December
OF LIPID
Reddy
and
8, 1989
SUMMARY: Experiments were undertaken to examine the effects of reduced (GSH) and oxidized (GSSG) glutathione on lipid peroxidation of rat liver Dependence on microsomal a-tocopherol was shown for the GSH microsomes. inhibition of lipid peroxidation. However, when GSH (5 mM) and GSSG (2.5 of lipid peroxidation was mM) were combined in the assay system, inhibition enhanced markedly over that with GSH alone in microsomes containing atocopherol. Surprisingly, the synergistic inhibitory effect of GSH and GSSG was also observed for microsomes that were deficient in a-tocopherol. These data suggest that there may be more than one factor responsible for the glutathione-dependent inhibition of lipid peroxidation. The first is dependent upon microsomal a-tocopherol and likely requires GSH for atocopherol regeneration from the a-tocopheroxyl radical during lipid The second factor appears to be independent of a-tocopherol peroxidation. and may involve the reduction of lipid hydroperoxides to their corresponding alcohols. One, or possibly both, of these factors may be activated by GSSG through thiol/disulfide exchange with a protein sulfhydryl moiety. Q 1990 Academic press, rnc.
Whereas breaking, its
interaction
are
less
a reduced
well
actually
exerts
reports
a-tocopherol
(3,5,8).
microsomes
a prooxidant
*To whom correspondence
system
and several
E by dietary
E. thus should
on lipid with
slowing
be addressed.
$1.50
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
that
inhibited
960
(3.5);
presence
effect
effect
of
of GSH
an additional was not
that
were
in this
peroxidation irreversible
of
lipid
indicated
This
- chain
peroxidation the
The inhibitory have
only
mechanisms
lipid
revealed
a microsomal its
not (1).
inhibit
or in microsomes
manipulation
effect
in association
of vitamin
that
(2-7).
- if
established
investigations
microsomes
denatured
GSH functioned
regeneration
Copyright All rights
liver
on microsomal of vitamin
constituents
Earlier
dependent
depleted
E as the major has been
(GSH)-dependent
in rat
in heat
0006-291X/90
membrane
glutathione
observed
that
of vitamin antioxidant
understood.
concentration
dependency
function
soluble with
peroxidation is
the
lipid
system (9).
protein
severely GSH
We proposed in
oxidation
the during
Vol.
166, No. 2, 1990
lipid
BIOCHEMICAL
peroxidation
suggesting
that
protect
its
factor
(3,5,9).
observed to act
as a free
radical
formation
(14).
specific
in
its
with
for
its
(9.15.16).
designed
to test
separately liver
peroxidation
reactive
the
and in
is
combination,
on NADPH-dependent
forms
extended
surprisingly
well
beyond
wherein that
EXPERIMENTAL
observed
was found
reduced
in which
GSH was
communication disulfide
lipid the for
were
(GSSG),
peroxidation lag
to
by
markedly
show a synergism
of glutathione
was
In association
assays
this
of GSH and glutathione
The results
and oxidized
in
was
compounds
none E.
GSH also with
(5)
thiol
as measured
formation, reported
microsomes
other
of vitamin
compared
or to
GSH-dependent
previously
of the
peroxidation
product
effects
liver
and cysteine),
a-tocopherol
The experiments
microsomes.
reduced
on lipid
(10)
the
or by preventing
reported
presence
by
of a-tocopherol
(6.12.13)
GSH because,
findings
studies, in rat
scavenger
in the
effect
in membrane
omitted
oxidation
dithiothreitol
RESEARCH COMMUNICATIONS
similar
In related
peroxidation
for
inhibition acid
decline
the
reported
peroxidation
requirement
inhibitory
thiobarbituric the
of lipid
(/?-mercaptoethanol,
be specific
have
(11). lipid
The inhibition tested
to delay
carrier
to inhibit
proposed radical
Others
GSH acts
cytosolic
AND BIOPHYSICAL
of rat
between
the
in lipid
GSH alone.
PROCEDURES
Animals and diets: Weanling male Long-Evans Hooded rats were raised from breeding stock purchased from Charles River Laboratories (Wilmington, MA) and fed chemically defined, torula yeast-based diets containing tocopherol stripped corn oil and lard as sources of fat (17). Rats were divided into two dietary groups: one group was fed a diet without vitamin E supplementation, referred to as vitamin E deficient, and the second group was fed the same diet supplemented with 150 IU vitamin E as dl-a-tocopheryl acetate/kg diet. Both groups of rats were fed their respective diets for 8 weeks. Prenaration of microsomes: The rats were killed by decapitation livers homogenized with 9 vol (w/v) of 0.15 M Tris-HCl. pH 7.4. for the preparation of microsomes have been described previously
and their Procedures (5).
Assay of lipid peroxidation: A modification (9) of the NADPH-dependent lipid peroxidation assay procedure described by McCay et al. (18) was used in these experiments. In brief, the reaction mixture contained 0.05 M Tris-HCl buffer, pH 7.4, 0.25 M NADPH, 0.012 mM FeSO4, varying concentrations of GSH and GSSG, and was incubated in a shaker water bath at 37O for the times indicated. The reduced and oxidized forms of glutathione were dissolved in water and the solution adjusted to pH 7.4 prior to their addition to the assay system. Lipid peroxidation was monitored by the formation of thiobarbituric acid (TBA)-reactive products that were quantified at 535 nm using an extinction value of 1.56 x 105M-1'cm-1 (19). Approximately 0.16 mg of microsomal protein/ml of reaction mixture was used in incubations for the determination of TBA-reactive products. Determination of GSH and GSSG: Concentration of reduced and oxidized glutathione was determined using a modification of the assay system described by Reed et al. (20). The HPLC was performed on a Beckman
961
system
Vol.
166,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
344 chromatograph equipped with a Beckman 165 W detector. A 250 mm x 4.6 mm DuPont Zorbax NH2 column fitted with a 50 mm x 4.6 mm Permaphase ETH precolumn was used for the analysis. The samples were eluted isocratically at a flow rate of 1.0 ml/min by a mixture of the solvents described in the original procedure and the eluent monitored at 365 nm. The microsomal lipid peroxidation assay system contained y-glutamylglutamate (y-GG) at a final concentration of 5 mMas an internal standard. The pH of the y-GG solution was adjusted to 7.4 prior to its addition to the reaction mixture and y-GG had no effect on the rate or magnitude of lipid peroxidation as measured by the formation of TBA reactive products. RESULTS The a-tocopherol concentration, determined as previously described (9). of liver microsomes used in these experiments was 0.35 k 0.03 and 0.01 -+ 0.002 ,ug/mg protein (x + SEM, n = 12) for rats fed the vitamin E supplemented and deficient diets, respectively. The effects peroxidation
of GSHand GSSG, separately
of liver
on lipid
microsomes prepared from rats fed the diet
supplemented with vitamin
E are shown in Fig. 1A.
alone produced a lag in lipid by TBA-reactive
or in combination,
peroxidation
product formation.
The addition
of > 30 min duration
In contrast,
the addition
of 5 mMGSH as measured of 2.5 mM
GSSGproduced no lag in peroxidation and was similar in reaction rate to that observed in the absence of both GSHand GSSG. The addition of 5 mM effect that GSHand 2.5 mMGSSGtogether, however, produced an inhibitory was synergistic.
In this case, the lag in lipid
peroxidation
was extended
greatly (approx. 90 min) compared with the addition of 5 mMGSHalone. Additional experiments (data not shown) demonstrated that the synergistic effect
of GSHin combination with GSSGwas concentration
example, increasing
the concentration
dependent.
For
of GSSGfrom 1.25 mMto 5 mMresulted
Additionally, increasing in increased increments in the inhibitory effect. the concentration of GSSGalone from 1.25 mMto 10 mMhad no effect on inhibition
of lipid
peroxidation
and mirrored the production
products obtained in the system containing
reactive
neither
of TBA GSSGnor GSH
(data not shown). The addition of either 5 mMGSHor 2.5 mMGSSGseparately to obtained from rats fed the vitamin E deficient diet failed to produce inhibition of lipid peroxidation (Fig. 1B). However, an inhibitory microsomes
effect
on lipid
peroxidation
was observed for microsomes from rats fed the
diet deficient in vitamin E in the presence of both 2.5 mMGSSGand 5 mM effect of GSHand GSSGcombined was heat labile since GSH. The inhibitory companion incubations (data not shown) using an ascorbate/ADP/Fe'+ no inhibition of dependent, nonenzymatic lipid peroxidation system revealed lipid
peroxidation in microsomes that were heat denatured. Changes in GSHand GSSGconcentration during the time-course in lipid
peroxidation
of rat liver
microsomes are shown in Fig. 2. 962
In incubations
Vol.
BIOCHEMICAL
166, No. 2, 1990
I
+E
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
A
Microsomes
60
-
E Microsomes
0
30
60
90
Time
Figure
over
both
time
GSH and GSSG (Fig.
was accompanied
GSH oxidation
occurred
presented
in Fig.
denatured
microsomes
There
observed
incubations that
the
this
system
obtained
net
because
in assays
absence
reduction
only between
a result
in GSH that in GSSG.
results containing
(data this
during
the
ZB).
These
GSH and GSSG in conversion
not
inhibition
heat shown).
system,
time-course data
those
to
either
of microsomes
occurred
Spontaneous
similar
of GSSG to GSH in
GSSG (Fig.
of the
decrease
increase
in GSSG concentration
synergism simply
in
or in the
containing
the
was not
2A).
by a concurrent
2A were
was no apparent
changes
180
Effects of GSH and/or GSSG on TBA-reactive product formation lipid peroxidation of liver during NADPH/Fe*+ -dependent microsomes from rats fed a diet supplemented (A) or deficient (B) in vitamin E. Each point represents a mean value from 8 Incubations were conducted with the following individual rats. -O-(0,0) : concentrations (mM) of GSH and GSSG. respectively: -A-(0,2.5): -A-(5,2.5). -O-(5,0);
1.
containing
150
120
(min)
nor
assay
indicate, of lipid
were
in however,
peroxidation
of GSSG to GSH.
DISCUSSION The present in extending fed
a diet
the
experiments lag
supplemented
in lipid with
demonstrate
a synergism
peroxidation
when liver
vitamin
E are 963
used.
between microsomes
The lag
period
GSH and GSSG from is
rats
Vol.
166, No. 2, 1990
extended
BIOCHEMICAL
approximately
3-fold
and cannot
be attributed
conditions
of the
the
observation
from
rats
that
independence
fed
suggests
microsomal
factor
the
deficient
peroxidation.
explain,
in part,
that
of Hill
and Burk
the
concerning
the
of liver
microsomes
from
consistently
detected
some GSSG in the
prior
lipid
approximately
peroxidation
5% contamination
than
rats
effects
I 30
60
.,
90
Time
120
,
,
150
180
E
labile,
E.
are
We have
prepared
amounts
may be even
GSSG y
and that
of GSH on lipid that
generally but
(3.5)
in vitamin
GSH
0
vitamin
of GSH and GSSG
reports
GSH solutions
GSSG (9)
was
microsomes
This
effects
This
interest liver
one heat
deficient
(
~~o---o---c-o 1
GSH alone
GSH and GSSG may also
inhibitory
assays. with
in E.
our previous
peroxidation to the
(13)
more
between
between
containing
Of particular present
inhibitory
The interaction differences
system
in vitamin
possibility in
RESEARCH COMMUNICATIONS
of GSSG to GSH under
was also
may be involved
on lipid
the
employed.
synergism a diet
with
conversion
procedures
this
were
compared
to any net
assay that
AND BIOPHYSICAL
to
greater
INACT;~IVZYME
y
GSH
ACTNE ENZYME (E-S-SO)
GSSG
(min)
Figure
2.
Time-course changes in GSH and bSSG concentration during NADPH/Fe'+-dependent lipid peroxidation of liver microsomes from rats fed a diet supplemented with vitamin E. Initial concentrations of GSIi(-0-) and GSSG(-•-) were 4.5 mM and 3.0 mM. respectively (A) and 0 mM and 2.6 mM. respectively (B). Each point represents a mean value from 8 individual rats.
Figure
3.
Proposed mechanism for synergistic microsomal lipid peroxidation. 964
GSsG
effect
of GSB and GSSG on
Vol.
166, No. 2, 1990
depending
upon
BIOCHEMICAL
the
quality
GSSG contamination peroxidation It
is
that
also
of lipid the
is
important
is in
low
Identification responsible
elusive.
Yonaha
glutathione
the
and Tampo
et al.
(23).
inhibition
1981 we reported
lipid
that
rat
GSTs (24).
Furthermore,
important lipid
role
peroxidation also
well
modification formation
has been
extent
that
occur. inhibition content
of membrane-bound
GSH-dependent.
have
heat
of a-
suggested
that
the
may be responsible by recent
for
peroxidation
cannot
completely
we suggested protection
that
its
ability
that
this
microsomal
moiety
(25).
is
associated system
membranes
out.
In
with
the
may play
from
lipid
GST is
damage
an
due to
hydroperoxides. activated
Indeed,
as a means of
This (22)
GSH-Trs
be ruled
enzyme
to reduce
the
effect. et al.
selenium-independent
which
of cellular
this
the microsomal
exhibit
activity
or
microsomal
of Haenen
a role
factor
has remained
for
reports
microsomes
labile
peroxidation
Nevertheless,
suggested
could
of GSH-dependent
of lipid
cysteine
event
of lipid
to the a-tocopherol
inhibition (21)
through
of the
a-tocopherol
microsomal
liver
established
In the inhibition
concentrations
(nonSe-GSH-Px)
in the
used.
of inhibition.
supported
of
peroxidase
small
(GSTs)
been
glutathione
is
the
has not
or Nagasaka in
for
S-transferases
suggestion
the
RESEARCH COMMUNICATIONS
substantial,
related
levels
of the
factors
that
positively
and even
result
is
of microsomal
to realize
microsomes
tocopherol
GSH preparation
GSH solution
independent
peroxidation
liver
of the
of the
AND BIOPHYSICAL
mixed
regulating
It
by covalent
disulfide
the microsomal
GSTs
(25-28). Based one factor
on our
results,
responsible
peroxidation.
for
The first
likely
requires
radical
during
appears
to be independent
semistable
fatty the
homolytic
both,
acid
rat
liver
shown
previously
TBA reactive lipid
microsomal (29)
peroxidation
was omitted. activity, peroxidation
factors
a protein between
assays It
markedly
was also reversed
shown
decyl-GSH.
that
for
the this
inhibitory
(29). 965
effect
of thus
or
This might inhibition of mechanism
and 2) reduced
to 02 consumption GSH compared
the
3. one,
moiety.
02 consumption
containing
reduction
by GSSG through
sulfhydryl
relative
GSTs)
alcohols,
in Figure
Support
1) reduced
the
the
by metal-catalyzed
GSH and GSSG in
peroxidation.
formation
the a-tocopheroxyl
radicals
may be activated
than
and
(possibly
corresponding
As shown
with
by
from factor
and may involve
propagating
synergism lipid
product
a-tocopherol
to their
of chain
may be more
upon microsomal regeneration
hydroperoxides
there
of lipid
The second
of hydroperoxides.
observed
that inhibition
of a-tocopherol
exchange
the
dependent
a-tocopherol
of these
thiol/disulfide explain
conceivable
peroxidation.
formation
cleavage
is
the GSH-dependent is
GSH for lipid
preventing possibly
it
ratio
in microsomal
to assays a potent
was
in which
inhibitor
of GSH on lipid
GSH of GST
of
Vol.
166, No. 2, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
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