Effect of doxorubicin-enhanced hydrogen peroxide and hydroxyl radical formation on calcium sequestration by cardiac sarcoplasmic reticulum

Effect of doxorubicin-enhanced hydrogen peroxide and hydroxyl radical formation on calcium sequestration by cardiac sarcoplasmic reticulum

Vol. 130, No. 2, 1985 8lOCHEMlCALAND8lOPHYSlCALRESEARCH COMMUNICATIONS Pages 739-745 July 31, 1985 EFFECT OF DOXORUBICIN-ENHANCED HYDROGEN PEROXID...

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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

All

Copyright 0 1985 rights qf reproduction

$1.50

by Academic Press, Inc. in an-v form reserved.

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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184-186. 7. 8. 9. 10. 11.

Moore, 131-138. Blayney, Noble, Okabe, Biophys. Okabe,

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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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