Vol. 130, No. 2, 1985
8lOCHEMlCALAND8lOPHYSlCALRESEARCH
COMMUNICATIONS Pages 739-745
July 31, 1985
EFFECT OF DOXORUBICIN-ENHANCED HYDROGEN PEROXIDE AND HYDROXYL RADICAL FORMATION ON CALCIUM SEQUESTRATION BY CARDIAC SARCOPLASMIC RETICULUM Richard Department
Received
of Medical
June
N. Harris
and James H. Doroshow
Oncology, City of Hope National Duarte, California 91010
Medical
Center,
19, 1985
This study investigated the effect of doxorubicin-related oxygen radical formation on Ca*+ uptake by rat heart sarcoplasmic reticulum vesicles. Enzymatic activation of doxorubicin by cardiac NADH dehydrogenase produced a dose-related inhibition of Ca*+ uptake that was enzyme- and cofactordependent and that was inhibited by catalase, various hydroxyl radical scaFurthermore inhibition of and the iron chelator deferoxamine %iee:i;ake paralleled the production of the hydroxyl radical by NADH dehydrogenase after doxorubicin treatment. These results suggest that doxorubicin-stimulated reactive oxygen metabolism can alter Ca*+ transport by cardiac sarcoplasmic reticulum and may represent one pathway involved in the cardiac toxicity of this potent antineoplastic agent. D 1985 Academic Press, Inc.
The clinical is
limited
cardiac
by the toxicity
hypotheses recent
exist
effect
Recently, cals (9,101.
the mechanism that
(SR)
previous
Furthermore,
major
site
several
investigators
1 Abbreviations used in the text: reticulum vesicles; .OH, hydroxyl
many different
Although
might
handling
exchanger
various
cardiac
drugin part,
of Ca2+ by
(3-6). demonstrated
that
any
reticulum by the oxygen
and skeletal
laboratories
(1,2),
be due,
sarcoplasmic
have found
of
may involve
of Ca*+ sequestration
by both from
(1).
have not convincingly
another
studies
form
that
by cardiac
(DOX)l
dose-dependent
cardiomyopathy
Na+/Ca*+
uptake
Ca*+ transport
doxorubicin
of DOX cardiotoxicity
on the
studies
of DOX on Cazt
however,
can disrupt
this
of the anthracycline
(71,
antibiotic
Ca*+ concentration
and the sarcolemmal
effect
antitumor
can be life-threatening
in cardiac
Unfortunately,
vesicles
of a cumulative,
have suggested
mitochondria
direct
production
to explain
changes
to a direct
of the
that
studies
related
usefulness
free
muscle
heart
(8).
radiSR
have shown that
DOX, doxorubicin; SR, sarcoplasmic radical; DMSO, dimethyl sulfoxide. 0006-291X/85 739
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Copyright 0 1985 rights qf reproduction
$1.50
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Vol.
130,
No. 2, 1985
certain
cardiac
flavin-containing
to a semiquinone to produce oxidizing study,
BIOCHEMICAL
free
superoxide species,
anion, the
we determined
enzymatically cardiac
MATERIALS
radical
activated
BIOPHYSICAL
dehydrogenases intermediate hydrogen
hydroxyl
whether
AND
radical reactive
00X could
(*OH) oxygen
alter
are capable
that
peroxide,
the
RESEARCH
reacts
with
COMMUNICATIONS
of activating molecular
and subsequently, (2,ll). metabolites
Ca2+ uptake
Thus,
oxygen potent
in the present
produced activity
the
DOX
by of
SR.
AND METHODS
The rat heart SR fraction was prepared at 4°C using cardiac tissue from 250-400 g Sprague-Dawley rats (Simonsen Laboratories, Gilroy, CA). The tissue was carefully washed and then homogenized as previously described (12) in iced 300 mM sucrose containing 40 mM Tris-histidine, pH 7.0. The homogenate was centrifuged at 4°C for 20 min at 1000 CJ; the supernatant was poured through 4 layers of cheesecloth and then centrifuged for 20 min at 8000 p. The resulting supernatant was centrifuged for 30 min at 45,000 9; the pellet was then resuspended in 10 ml of 600 r&l KC1 and 40 mM Tris-histidine, pH 7.0, and centrifuged for 30 min at 45,000 The final pellet was resuspended in 300 mM sucrose containing 10 mM imi 3 azole, pH 7.0. Protein was determined by the method of Bradford (13). SR was either used fresh or kept frozen at -1OO'C and used within the week. Uptake of Ca2+ by SR was measured by dual wavelength spectrophotometry using arsenazo III as the indicator (14). In brief, 150 pg samples of cardiac SR protein were added to the Ca2+ uptake buffer (100 mM KCl, 10 mM oxalate, 10 mM Hepes, pH 7.0) which contained 8 mM ATP, 10 IIW'IMgCl , and 1 n@i sodium azide, III in a final volume of 3 ml. After a 4 min 20 pM Ca2+ and 30 pM arsenazo incubation at 37"C, the change in absorbance at the wavelength pair 675 vs 700 nm due to the addition of 90 nmol of EGTA was measured using a Shimadzu UV-3000 spectrophotometer. Relative Ca2+ uptake was determined from a standard curve (change in absorbance at 675/700 nm vs change in Ca2+ concentration) by sequential additions of 6 nmol of EGTA to the complete 3 ml mixture containing heat-denatured SR. Exposure of SR to various treatments before measurement of Ca2+ uptake was performed at 37°C in a shaking water bath in I50 mM sucrose, 5 i@l imidazole, 50 mM KCl, 5 mM oxalate, 5 ti Hepes, 5 mM MgC12, 0.5 mM sodium azide, 5 mM KCN, and 20 mM potassium phosphate, pH 7.5, in a total reaction mixture of 1 ml containing 200 pg of SR protein and experimental reagents where indicated. The control rate of Ca2+ uptake by fresh SR (no incubation at 37°C; mean t S.E.) was 270 t 13 nmol/4min/mg SR protein (n = 10). This rate was not changed significantly by any of the free radical scavengers used in these experiments. Ca2+ uptake by SR alone incubated at 37°C for 30 min (238 f 16; n = 14) or 45 min (227 f 34; n = 3) was not significantly different from the rate of Ca2+ uptake by SR unexposed to incubation at 37°C. All results in this study were expressed based on concurrent, daily control experiments. NADH dehydrogenase activity and .OH production (assayed as the evolution of methane from dimethyl sulfoxide) were measured as described previously (2) except that experiments were performed in the buffer used to expose SR to uox. DOX hydrochloride was purchased from Adria Laboratories, Columbus, OH; 5-iminodaunorubicin was obtained from the Drug Synthesis and Chemistry Branch, NCI, Bethesda, MD. Arsenazo III and NADH dehydrogenase were purchased from Sigma Chemical Co., St. Louis, MO. Catalase 1.1 x 106 units/ml devoid of superoxide dismutase activity was obtained from Buehringer IN and was dialyzed once against Mannhein Biochemicals, Indianopolis, 740
BIOCHEMICAL
Vol. 130, No. 2, 1985
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
saline before use. the data were analyzed
phosphate-buffered mean f 1 S.E.; means (15).
All values are expressed as the by the 2-tailed t test for independent
RESULTS Previous
studies
from
by NADH dehydrogenase .OH formation
(2).
revealed
for
that
or NADH alone more,
these
on the
our
in the presence Detailed
the
agents
experiments
concentrations
either
shown
effect alone
have shown that of NADH results
control
had no direct
absorption
laboratory
III-Ca2+
Table Effect
of
Experimental
Control
Enzymatically
complex
(data
Activatd Doxorubicin by Rat Heart SR
on Ca*+
(40 nmol)
(11,000
;; ;
97 70 41 71 80 79
units)
(40
(11,000
units)
$J-
mean
f S.E.
of
3 to
2d Es 9c 7d 7d ld
a7 +
2d
from
control,
p < 0.01.
dsignificantly doxorubicin,
different p < 0.05.
from
complete
reaction
by boiling 741
for
3d
"Materials and Methods" The control rate of The final reaction of NADH dehydrogenase.
5 experiments.
different
heat-inactivated
t f f + + f
88 f
nmol)
csignificantly
were
Uptake control)
77 -r 2d 38 2 12c
aThese experiments were performed as described in in a volume of 1 ml incubated for 30 min at 37Y. Ca*' uptake was 238 f 16 nmol/4 min/mg, n = 14. mixture contained 5 umol of NADH and 220 milliunits the
Uptake
90 i 5d 90 + 13d
LLIminodaunorubicin
are
shown).
100
+ urea (50 umol)
eEnzymes
not
Ca*+ (% of
heat-inactivated catalase deferoxamine (100 nmol) N-acetylcysteine (5 umol) glutathione (5 pmol) histidine (15 pmol) dimethylurea (5D pmol)
bvalues
SR; further-
1
- doxorubicin - NADH - NADH dehydrogenase Using heat-inactivated NADH dehydrogenasee + + + + + +
investigation
had no significant
alone)
Doxorubicin
+ catalase
current
by cardiac
System
(SR
iron-dependent
I DOX, NADH dehydrogenase,
in Table
or in combination
of the arsenazo
in
in the
on Ca2+ uptake
DOX metabolism
system 10 min.
containing
effect
Vol. 130, No. 2, 1985
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
3%= pco.05 c*= pco.01
0
I
I
I
10
20
30
DOXORUBICIN
I
40
(PM)
Effect of doxorubicin on Ca2+ uptake by rat cardiac SR. F- R (200 pg) in a 1 ml volume were exposed to NADH (5 umol), NADH dehydrogenase (220 milliunits) and various concentrations of doxorubicin for 45 min at 37°C; Ca2+ uptake at each doxorubicin dose was determined in triplicate as described in the "Methods."
However,
as illustrated
reaction
system
Inhibition
including
inhibition
ficant Inhibition level are
of Ca2+ uptake
entirely
consistent
shown
and enzyme
3.
10 min
of
This
linear
decrease
the
incubation
by the presence
at
doxorubicin
37OC and
and
1 demonstrates
(Fig.
2). that
DOX treatment
These oxygen
signi-
1 10 pM.
related
varied
that
with
at DOX levels
finding
Ca2+ uptake
reduction
increase
NADH dehydrogenase,
to the experiments
radical directly
with
(21. of
in the function
coincident of
after
inhibition
Fig.
of the
was dose-dependent
study
our previous
A significant
DOX, intact
DOX dose was also
used in the
complete
the
each component
occurring
employed
SR to
by 67% (P < 0.01).
Furthermore,
at a constant
levels of
only
explained
including
by doxorubicin
with
course
in Fig.
SR required
I).
by NADH dehydrogenase
drug
of cardiac
Ca2+ uptake
in Ca2+ sequestration
of NADH dehydrogenase
The time
in
(Table
of Ca2+ uptake
production the
system
cofactor
reductions
exposure
in cardiac
drug metabolizing
and the reduced
I,
DOX reduced
of Ca2+ uptake
enzymatic
the
in Table
was
in Ca2+ uptake nearly
of the in cardiac
742
by DOX-treated occurred
complete
after
Ca2+ pump with
*OH production SR.
time
SR is after 45 min.
may be
by NADH dehydrogenase
Vol.
130,
No. 2, 1985
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
** = P
I 110
1 0
I 220
NADH
I 360
DEHYDROGENASE
1 440
(mu)
2. Effect of NADH dehydrogenase activity on doxorubicin-dependent inhibition of Ca2+ uptake by cardiac SK. These experiments were performed in triplicate at 37°C for 30 min using 5 pmol NADH, 0 to 440 milliunits of NADH dehydrogenase, 40 nmol doxorubicin, and 200 pg of SR protein.
Fig.
Because could
be
DOX,
this
system.
peroxide,
but
cardiac
experiments
inhibited
activated in
these
SR.
by
an
oxygen
we examined
the
As not
shown
the
in
suggested
that
radical
cascade
effect Table
of 1,
the
several
free
in by
heart
SK
enzymescavengers of
significantly
scavengers
rat
radical
a scavenger
enzyme .OH
uptake
produced
catalase,
heat-inactivated
Furthermore,
Ca2+
hydrogen
protected
-N-acetylcysteine,
glutathione,
-1
/
0
/
I
10
20
INCUBATION
Fig.
3.
Time-course
of
I
/
TIME
doxorubicin-dependent
4’ 5
30
(min)
inhibition
of
Ca2+
uptake
by cardiac SR and hydroxyl radical production by NADH dehydrogenase. Conditions for the Ca2+ uptake experiments were the same as those shown Fig. 1 except that 40 pM doxorubicin was used. Hydroxyl radical production in the presence of 100 mM DMSO was assayed in triplicate under identical conditions including the addition of 200 pg/ml of SR protein.
743
in
Vol. 130, No. 2, 1985
histidine, free
and dimethylurea
radical
production
structurally
similar
tive
effect
that
is
(161,
BIOCHEMICAL
for
significantly
free
radical
cant
effect
the
at equimolar
formation
agent
which
effect
moiety
by cardiac
of
the
had no protec-
iron
in a form reactions
of DOX-stimulated
concentration, quinone
effect
hand,
urea
chelates
oxygen
radical
5-iminodaunorubicin, so that
by NADH dehydrogenase
on Ca2+ uptake
toxic
in oxidation-reduction toxic
at the
the
On the other
*OH trapping
participation
of DOX modified
late
reduced
Deferoxamine,
reduced
Finally,
analogue
ineffective
system.
unavailable
significantly
by DOX on Ca*+ uptake. but
in this
metabolism.
all
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
it
(2),
SR (Table
an
does not
produced
stimu-
no signifi-
1).
DISCUSSION In these
experiments,
significantly
inhibited
that
the time
course
.OH,
and that
this
trapping bolism
of altered
As we (2,12) is reduced
transport
chain,
probably
the
Furthermore,
our
results
Although directly scavengers
ability
the
addressed
the free
DOX quinone
by various reactive
of
radical
oxygen
meta-
that
the
precise
prior mechanism
in this
study,
our
flux
inability
of previous
744
of the
at this
to the
site.
has not
been
thiol-containing
in ameliorating are related
by
investigators
in SR may be related
Ca2+ uptake
findings
produced
Ca2+ homeostasis.
for
efficacy
complex.
pathophysiological
injury
and glutathione that
radical
to membrane
the
electron
NADH dehydrogenase
cardiac
altered
the DOX
mitochondrial
may have direct
of DOX on Ca2+ uptake
activation
suggest
of the
of SR to maintain
-N-acetylcysteine
Ca2+ pump might
the production
have reported,
of the cardiac
that
indicate
an effect drug
(17)
portion
suggest
to the
for
for
paralleled
drug-induced
oy a component
here
consequences
necessity
The observation
or abolished
investigators
at an early
of electrons
to demonstrate
that
SR.
DOX
our findings.
reported
diversion
cardiac
be reduced
suggests
and other
quinone
by rat
enzymatically-activated
Ca 2+ transport
could
strongly
explain
Our results
Ca2+ uptake
effect
agents, could
we have shown that
damage to the
to a free
radical-
8lOCHEMlCALANDBlOPHYSlCALRESEARCH
Vol. 130, No. 2, 1985
related
oxidation
of cardiac shown
that
in the
regard
in heart explain
antitumor
Ca*+ uptake
that
previous
agents,
toxicity
--in vivo
SR. at least
fashion This part
(2)
oxygen
In any event,
leads
cardiac
it
*OH in an
to the
radical-related
of the
have
-N-acetyl-
can produce
which
system
studies
including
(18).
by NADH dehydrogenase
peroxide-dependent
could
groups
in this
DOX cardiac
of Ca*+ uptake
anthracycline
of note
DOX metabolism
and hydrogen
in Ca*+ uptake
is
sulfhydryl
of sulfhydryl-containing
in preventing
seems clear
bition
It
SR (8).
the effectiveness
cysteine,
iron-
of critical
COMMUNICATIONS
inhi-
alteration toxicity
of the
agents.
ACKNOWLEDGEMENTS We wish to thank Sunny Ilagan for her help in the preparation manuscript. This stud-v was supported bv orant CA 31788 from - the Leukemia Society of America:
of the the NC1 and by
REFERENCES 1. 2.
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Lenaz, L., and Page, J. (1976) Cancer Treat. Rev. 3, 11 l- 120. Doroshow, J. (1983) Cancer Res. 43, 4543-4551. Olson, H.M., Young, D.M., Prieur, D.J., LeRoy, A.F., and Reagan, R.L. (1974) Amer. J. Path. 77, 439-454. Villani, F., Piccinini, F., Merelli, P., and Favalli, L. (1978) Biochem. Pharmacol. 27, 985-987. Revis, N., and Marusic, N. (1979) Exp. Mol. Pathol. 31, 440-451. Caroni, P., Villani, F., and Carafoli, E. (1981) FEBS Letts. 130,
184-186. 7. 8. 9. 10. 11.
Moore, 131-138. Blayney, Noble, Okabe, Biophys. Okabe,
L.,
Landon,
E.J.,
and Cooney,
D.A.
(1977)
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18,
L. (1983) in Cardiac Metabolism (Drake-Holland, A.J. and M.I.M., eds) pp. 19-47, John Wiley and Sons, New York. E., Hess, M.L., Oyama, M., and Ito, H. (1983) Arch. Biochem. 225, 164-177. E., Hiyama, E., Oyama, M., Odajima, C., Ito, H., and Cho, Y. (1982) Pharmacology 25, 138-148. Bachur, N.R., Gordon, S.L., and Gee, M.V. (1978) Cancer Res. 38,
1745-1750. Doroshow, J.H. (1983) Cancer Res. 43, 460-472. 13. Bradford, M. (1976) Analyt. Biochem. 72, 248-254. Scarpa, A. (1979) Meth. Enzymol. 56, 301-338. :45: Armitage, P. (1971) Statistical Methods in Medical Research, pp. 104-126, Blackwell Scientific Pub., Oxford. 16. Rosen, H., and Klebanoff, S.J. (1981) Arch. Biochem. Biophys. 512-519. Thayer, W.S. (1977) Chem.-Biol. Interact. 19, 265-278. Doroshow, J.H., Locker, G.Y., and Myers, C.E. (1981) J. Clin. 12.
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745
208, Invest.