Document 6bevn9gvy6gkJGdObZjzxmZm1
Vol. 148, No. 1, 1987 October 14, 1987
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages X3-160
INCFUWSEDEFFLUXRATHERTBANOXIDATIONISTHEMECHANISM
OF GIUl?ATHIONJZ DEPLEMDN BY l-MEIWLr4-PHENYL-1,2,3,6-
PYlUDINE(MPIlJ)
DonatoDiMonte,MarthaS.
Sandy, andMartynT. Smith
Dapartnentof Biomedical andEuvironmentalHealthSciences, School of Public Health, University of California, Berkeley, CA 94720
Received August 31, 1987
SJMM?GtY. Incubation of isolated hepatocytes in the presen ceofeitherthe
parkihsonian-inducing
cqmmd 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP) or its putative toxic metabolite l-methyl-4-phmylpyridinim
ion (BPP+)
led to a depletion of intracellular
reduced glutathione (GSH), which was mostly
recovered as glutathione disulfide (GSSG). However, both MFTP- and MPP+-induced
glutathione perturbances were relatively unaffected by the prior inhibition of
glutathione reductase with 1,3-bis(2-&loroethyl)-l-nitrosourea
(BCNLJ),
suggesting that intracellular
oxidation was not the major mchani 'Sal involved in
theGSH1os.s. Inclusion of cystine in the incubation mixtures revealed a time-
deperdent formation of cysteinyl glutathione (WSG), indicating that an
increased efflux was mostly responsible for the MPTP- and MPP+-induced GSH
depletion. Therefore, the masurementofGSSG,tichisapparently
formed
extracellularly,
was not associated with oxidative stress. 0 1987 Academic Press, Inc.
The possible generation of oxygen radicals and their role in cell damage
caused by the parkinsonian-inducing
mmpound MPTP (I-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine)
(1) hasbeenthe subjectofmuchcontrmersy.
Reduced
glutathione (GSH) is involved in a variety of detoxication reactions against
oxidizing species produced during the metabolism of xenobiotics (2). Therefore,
a selective depletion of GSH in the substantia nigra of mice injected with MPTP
has been interpreted as an index of regional vulnerability to oxidative stress
(3). A lower concentration of GSH has also been found selectively in the substantia nigra of patients with idiopathic Parkinson's disease (4), leading to
thepossibility
0fGSHconsmptionbeingamoregemra
leventinthe
pathological processes occurring in this region of the brain.
Several lines of evidence point to the fully oxidized metabolite l-methyl-
4-phenylpyridinim
ion (MPP+) as the ultimate mediator of MPTP-induced cell
damage (6,7). concentrations
Johannessen et al. (8) havemmsuredan increase intheplasma of oxidized glutathione (GSSG) after systemic administration of
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Vol. 148, No. 1, 1987
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Mm+ tc rats, suggesting that this czqwud induces oxidative stress in vivo.
Recent work in cnr labomtory using isolated rat hepatocytes as an in vitro
modelhasshawn,hawever,thatbothMprpand~P+areunlikelytoproduce
general cytotoxicity
as a consequence of oxygm radical generation, even if both
mmpomds cause a depletion of intracellular GSH (9,lO). Here, we repm-t that
the GSH depletion induced by both MPIP and MPP+ is not due to intracellular
oxidation, but rather to an increased efflux of the reduced tripeptide frm the
cellandits
subsequent oxidation intheextracellularspace.
MA- AND IGTl?HOE
MPTP (hydrochloride salt) and MPP+ (iodide salt) were purchased frcnn Re-
search I&chemicals (Wayland, MA). Collagenase (grade II) was from Bcehrirqer
(Mannheim, West Germany). BcNuwas~ysuppliedbytheDrugSynthesisand
Chemistry Branch, Division of cancer Treatsant of the National Cancer Institute,
Bethesda, MD. g body weight)
Hepatocytes and incubated
were (10
8'soclealtlesd/ml)
frm male, Sprague-Dawley rats (220-280 in Krebs Hmseleit buffer (11).
Cell viability was assess& as exclusion of Trypan blue (11) and was always
greater than 90% at the bzginning of the viments.
When inhibition of
glutathione reductase (EC 1.6.4.2) was desired, the cells were pretreated for 20
min with 1,3-bis(2-&loroethyl)-1-nitrosour
ea(EKNU)andthenincubatedfor75
min in fresh media supplemented with 1 m methionine as in (12). Glutathione
reductase activity was measured spctrophotcrmetrically
by monitoring the
oxidation of NADPH (at 340 nm) in the pre.smceof50mGSSG,0.1llMNADmand
1% Triton X-100. Orily hepatcxzyte preparations with > 90% inhibition were used
intheexperimentsshownhere.
To seasure GSH efflux frm hepatocytes, 0.2 EM
cystinewas included inthe incubationmixtures
as in (13). GSH, GSSGandthe
cysteinylglutathione
disulfide (QSSG) were detectedby HPLC as described in
(14). At the indicatedtimes,
samples (lml) were taken andthe cellswere
immediately separated frm the media by rapid centrifugation (3 s at 13,000 g)
in a microfuge (Eppendorf 5415). Whencystinewaspresentinthe
incubationthe
cellpelletswerewashedonceandthen
resuspended inbuffer.
GSHwasextracted
by addition of 70% perchloric acid (1:20, v/v).
Incubation of isolated hepatocytes in the presence of 1.0 mMMPTP led to a
progressive decline in the intracellular
level of GSH (Figure lA). The initial
GSH concentration was 38.5 _+ 3.9 nmles/106 cells ahd 29.5 _+ 2.7 moles were
lost after 80 min incubation, prior to the occurrence of detectable sighs of
hepatocytedeath (datanotshown).
Approximately half of this decrease in GSH
occurred by 40 min. At this tima point 92% of the missing GSH was recoveredas
GSSG (intracellular+
extracellular).
The role of H202 generation in the oxidation of GSH during MPIP exposure
was investigated by inhibiting glutathione reductase activity with I3cm and,
154
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
i ----a---- I
A IL
11 20 40 60 8(
Time (mid
GSHdepktionmdGSSG formtioncausedby1.OnM
MPlPin
untrmed (A) andE!CNJ-treated (B) hepatooytes. Fkeshlyisolated
hepatooytes were either pretreated with EKNU (filled symbols) or
notpretr&zed (opensylkols) andthen~to
l.ommTP
(0) or m addition (0). Atthetimepointsshown,GSHandGSSG
oontentswereanalyzedascksoribedinMethcds.
Thedata
represent the mean (2 S.D.) of 4 separate experiments.
thus, impairing the action of glutathione peroxidase (12). As previously re-
ported (9), KNM.reat& hepatocytes were not significantly more susceptible to
the toxic effects of MPlT than control cells. FigurelBshowsthattherateof
GSH loss caused by MPTP was not significantly altered by the inhibition of
glutathionereductase.
Approximately 50% oftheGSHwaslost40minafterMPrP
addition, similar to the rate of loss in untreated hepatocytes. The initial GSH
concentration was 51.1 _+3.9 nmoles/106 cells and the intracellular value fell
to 8.7 f 3.1 moles after 80 nun. The overall GSH decrmse was, therefore,
guantitatively greater than that measured in untreated cells, but this
difference was much less than that expeckd for a potent H202-generating
aampound (12) * TherecoveryofGSHasGSSGinFigure
lBwas88%at4Omin.
FYm the data shown in Figure 1, one can therefore conclude that the inhibition
of glutathione redu&ase does not affect the rate of GSH oxidation caused by
METP, suggesting a minimal role for H202. Direct inhibition of the enzyme by
MPTPcouldexp1ainthisphe.n
cmenon, but the maximal activity of glutathione
reductase inthehepatocyteswas
foundtobeunaffectedbyMPrPexposme
(data
not shown). The possibility that GSH oxidation ocmrred intheextracellular
1.55
Vol. 148, No. 1, 1987
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Tr
40
L
p ($D 30 0r
G 5E 20
.z
z
E 10
T
0 -L
20 40 60 80
Time (min)
Fig. 2. mt
of MPlT-induced GSH efflux by addition of cystine to
the hepatocyte suspension. IrmtdionswereprfonneA
in the
presence of 0.2 nM cystine. Atindic&?dtimes,inbacellular
GSH (O), extmcellularcyssG (H) aIYAtotal (intracellular+
extEloellular) GSSG(d) were mElsurd as de5crm in Methods.
l'he *cellular
GSHcontent of the control cells was 38.8k3.3
mml/lO cells and was little affected by incubation for 80 min.
Ears represent the mea@.D. for 4 separate cell preparations.
space therefore seemed likely. To test this hypothesis, hepatocytes were incubated in medium supplemented with 0.2 mM cystine and the formation of cysteinyl glut&Cone (CySsG) determined as an index of GSH release from the cells (13).
Figure 2 shawsthatatime-dependentformationof
CySSGwas observed
after addition of MPTP to hepatocyte suspensions inthepresence
of cyst&e.
oxidationof GSSGaccounted foronlyabout7%
ofthetotal
GSHdecrease in
these experiments, while 84% of the GSH was recoveredasCySSGat8Omin.
Iess than 1.5% of the CySSGwas faund intracellularlyatalltimepoints
(data not shown). Incontrolcells, 3.4 f 0.9 to 6.2 _+ 0.5 moles/ml
the mncentmtion of CyS.SGrcse from after lhour incubation, in agreemen t with
previously reported data on GSH efflux fmn freshly WhenMFTPwasaddedtoJXNJ-treatedhepatocytesinthep~ the loss of GSH was, indeed, due mostly to oxidation
isolated (Figure
hepatocytes (13). ofcystine,
3). Total GSSG
156
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
50
p 40
$ co 07 . 30 5 E 5 3 '5 20
$
I1 T
5 IO 0-
1
40 60
Time (min)
Fig. 3. Effect of cystine on glutathione measurement in Km-treated
hepatocytes. FYe&lyisolakdhepatcqteswerepreincuba~as descriJxdinMethc&withBcNuandthenexpceedtol.OnMMprpin
the presence of 0.2 lml qstine.
Atthetimepoints
indicated,
intra~l.lularGSH
(a), exbacellularQSSG
(a) andtotal GSSG
(intrac`sllular, q + extracellular,~
) weremeas~&byhigh
mozn!ance liquid Ftogmphy
(18). In cxrltil cells, GSH
values (51.2 nmol/lO cells at 0 min) did not change
significantly
during 80 min incubation and the co ncentration of
CyssG -by
3.2kO.7 mmles between 0 and 80 min. Bars
represent the -S-D.
for 4 separate expxin-ents.
accounted for 57% arCi CySSG for 24% of the difference between GSH concentrations
at time 0 and after 80 min incubation.
The role of oxidative stress in the depletion of GSH induced by the puta-
tive toxic n&abolite of MPTP, MPP+, was then assessed in isolated hepatocytes.
Theresults reported inTable I showthat2.OmMMPP+caused
a 40% decrease in
the intracellular
GSH level after 80 min incubation. Formation of GSSG, which
accounted for approximately 80% of this loss, was not due, hmever, to intracel-
lular H202 generation. Whencystinewasaddedtothe
incubation,
therecovery
of GSHas CySSGandGSSGwas 76% and 5% respectively after BOmin, indicating
that an increasedeffluxof
GSHwasresponsible fortheGSHloss
inducedby
?QP+, as well as for that observed with MPIP.
157
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABIEI
EFFEcrOF2.O~~+oNHEpATo(NTEG~ONEINTHEABsENcE ANDPRFSENCEOFCYSMNE
Min. Treaw
None cyst&
MPP+ cystine + MPP+
GSH 37.e3.8
0 GSSG
2.9kl.l
cys= 0.5co.2
38.4k4.0 l.RO.4 3.1i1.4
36.e3.1 3.220.9 0.20.2
37.2k3.5 1.8f0.5 3.621.1
GSH 36.924.3
40 GSSG
2.8_+0.7
cys= 0.7kO.3
GSH
37.124.2
80 GSSG 3.3k1.2
1.420.6
36.7k3.4 1.eo.9 4.221.3
37.8k4.0 2.320.5 6.4k1.3
28.w2.9 9.e2.1 1.420.5
21.8k2.2 15.2k2.7 2.2kO.3
27.23.8 2.220.7
10.8f1.7
20.323.6 2.7fl.O 16.5g.8
Incubtionswereperfom&intheabsence
irdicat&.
IntmzenllarGSH,
extracel1ularcySSGwere-
(exprssed
as GSH or GSH
separate preparations.
e&v./10
or presnce total (inticellular+
of cystine ex-tmcellular)
(0.2 nM) as 6SSGand
y~~rding cells)
to Rexi et al. (18). Each value
rep resentsthemean
(+S.D.)
of3
DISCUSSION
The data presented in this study allow a detailed analysis of the
biochemical events followingexposureofhepatccyte.stoMprp,
andsupportour
recent conclusion that oxidative damage does not play an important role in the
general cytotoxic effects of this compound (9). Hz02 dces seem to be foxmed
durirq MPI!P metabolism in hepatocytes, probably as a prcduct of the oxidative
reactionwhichis
c&alyzedbymmamine
oxidase intheoutermembraneof
mitochondria, gemaratirq the 2,3-dihydropyridinium
derivative of MFTP (5). The
rate of H202 formation does not seem to ovemhelm the capacity of the cell to
scavengethis oxidant, however, arddidnot induce apparentbicchemicaleffects
(e.g., GSH oxidation) unless glutathione reductase activity was inhibited.
The GSHloss inducedbyMprphas
conseguentlykenshownto
involve two
different mechanics, oxidation and efflux, in both control and EKTW-treated
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
hepatocytes. Thebal~~between~~ofthesetwo~ismspredaminatesis
dependent primarily on the activity of the glutathione peroxidase/glutathione
reductasesystem.
When this system is not compromised, no oxidative processes
seemtoba involved in the intracellular
loss of GSH causedbyeither
MPTP or
MPp+. This effect is due rather to an increased efflux of GSH with GSSG being
formed subseguently by oxidation in the extracellular space. Therefore, no
relationship links the measuremen t of GSSG after hepatocyte exposure to MPIP
with the generation of oxygen radicals.
Ihelossof GSH inducedby~P~was~~sl~~thanthat~~~with
MPIP (even at twice the concentration), reflecting the limited access of this
chargedm&aboliteto
thecell (7), andsuggestingt.hattheGSHefflux
induced
bythesepyridineccxnpoundsistriggeredby
intracellularevents.
The
biochemicalmechanismunderlyingtheseprocessesisthe
subjectofongoing
studies inourlaboratory.
GSH might be released and oxidized extracellularly
following the breakdown of an unstable glutathione conjugate excreted from the
cells (15). This hypothesis, however, is unlikely, since neither MPIP nor MPP'
have been found to react with GSH and form a glutathione conjugate. A more
likely explanation is based on the fact that GSH is negatively charged within
cells and perhnbations of membrane potentials can influence its efflux (16,17).
Both MPIP and MPP+ toxicity in hepatocytes seem to be correlated with an earlier
rapid depletion of ATP (18). The relationship between these toxic events and glutathione status might provide the key to fully interpret the phenomenon
described inthis study. In accordance with this hypothesis, preliminary
results show that other wunds which perturb cell membrane potentials (e.g.,
valinamycin) and/or impair the supplies of cellular ATP (e.g., antimycin A) also
deplete intracellular
GSH via a mechanism of increased efflux (D. Di Monte, M.S.
Sandy and M.T. Smith, manuscript in preparation).
A-. SupportedbytheHealthEffectsCcrmponentoftheUniversityof
CaliforniaToxicSubstanaes
Program (D.D.), the Northern California Occupational
Health CXnter (M.S.S.) and the National Foundation for Cancer Research (M.T.S.).
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