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

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.

Burton, G. W., Joyce, A., and Ingold, K. U. (1983) Arch. Biochem. Biophys. 221. 281-290. Christophersen, B. 0. (1968) J. Biol. Chem. 106, 515-522. Reddy, C. C.. Scholz, R. W., Thomas, C. E.. and Massaro, E. J. (1982) Ann. N.Y. Acad. Sci. 393. 193-195. Burk, R. F. (1982) Biochem. Pharmacol. 31, 601-602. Reddy, C. C., Scholz, R. W., Thomas, C. E., and Massaro, E. J. (1982) Life Sci. 31, 571-576. Burk, R. F. (1983) Biochim. Biophys. Acta 757, 21-28. A. I. (1986) Biochem. Pharmacol. 35, Belouqui, O., and Cederbaum, 2663-2669. Wefers, H., and Sies, H. (1988) Eur. J. Biochem. 174, 353-357. Lipids 24, Graham, K. S., Reddy, C. C., and Scholz, R. W. (1989) 909-914. Robey, S., and Mavis, R. (1986) Fed. Proc. 45, 1671. Behrens, W. A., and Kaasgaard. S. (1986) Fed. Proc. 45, 1809. Burk, R. F., Patel, K., and Lane, J. M. (1983) Biochem. J. 215, 441-445. Hill, K., and Burk, R. F. (1984) Biochem. Pharmacol. 33, 1065-1068. Haenen, G. R. M. M.. and Bast. A. (1983) FEBS Lett. 159, 24-28. Murphy, M. E., and Kehrer, J. P. (1987) J. Chromatogr. 421. 71-82. Murphy, M. E., and Kehrer, J. P. (1989) Arch. Biochem. Biophys. 268, 585-593. Meeker, H. C., Eskew, M. L., Scheuchenzuber, W., Scholz, R. W., and Zarkower, A. (1985) J. Leuk. Biol. 38, 451-458. McCay, P. B.. Gibson, D. D., and Hornbrook, K. R. (1981) Fed. Proc. 40, 199-205. Buege, J. A., and Aust. S. D. (1978) In Methods in Enzymology (S. Fleischer and L. Parker, Eds.), Vol. 52, pp. 302-310, Academic Press, New York. Reed, D. J., Babson, J. R., Beatly, P. W., Brode, A. E., Ellis, Anal. Biochem. 106, 55-62. w. w.. and Potter, D. W. (1980) Yonaha. M., and Tampo. Y. (1986) Chem. Pharm. Bull. 34. 4195-4201. Haenen, G. R. M. M.. Tai Tin Tsoi, J. N. L.. Vermeulen, N. P. E.. (1987) Arch. Biochem. Biophys. 259, Timmerman. H., and Bast, A. 449-456. Nagasaka. Y., Fujii, S., and Kaneko, T. (1989) Arch. Biochem. Biophys. 274, 82-86. Reddy, C. C., Tu, C.-P. D.. Burgess, J. R., Ho, C.-Y., Scholz, R. W., and Massaro. E. J. (1981) Biochem. Biophys. Res. Commun. 101, 970978. Morgenstern, R., DePierre, J. W.. and Ernster, L. (1979) Biochem. Biophys. Res. Commun. 87, 657-663. (1986) Biochem. Pharmacol. 35, 435-438. Masukawa. T., and Iwata, H. Arch. Biochem. Biophys. 270, M. W. (1989) Aniya. Y., and Anders, 330-334. J. Biol. Chem. 264, 1998-2002. M. W. (1989) Aniya, Y., and Anders, Scholz, R. W., Graham, K. S., Gumpricht, E., and Reddy. C. C- (1989) Ann. N.Y. Acad. Sci. 570, 514-517.

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