HEMOGLOBIN, 4 ( 5 & 6 ) , 735-745 (1980)

THE EFFECTS OF ADRIAMYCIN (DOXORUBICIN H C L ) ON HUMAN RED BLOOD CELLS

K . Shinohara and K.R. Tanaka

Department of Medicine Harbor-UCLA Medical Center, Torrance, CA 90509

ABSTRACT

We have studied the e f f e c t s of adriamycin (doxorubicin H C 1 ) The peroxidizing e f f e c t of adriamycin on human red blood c e l l s .

on the t h i o l s of red ce l l cons t i tuents resu l ted i n decreased glutathione s t a b i l i t y , and oxidation of hemoglobin and membrane protein components 1 , 2 , and 3 , forming la rge molecular weight complexes. Membrane l i p i d s were a l so peroxidized. Adriamycin i t s e l f d id not i n h i b i t the enzymes of t h e reductions system (91 ucose-6-phosphate dehydrogenase, 6-phosphogl uconic dehydro- genase, glutathione reductase, glutathione peroxidase, ca t a l a se , superoxide dismutase) of the red c e l l s . the potential of inh ib i t ing ATPase, including b o t h Na-K-dependent ATPase and ouabain insensitive ATPase, a t concentrations not in- h ib i tory t o o ther enzymes, the net sodium content increased, and potassium content decreased a f t e r incubation of red c e l l s w i t h adriamycin a t high concentrations. The experimental r e su l t s described w i t h adriamycin may serve as a model f o r t he possible mechanism of card io toxic i ty observed i n i t s c l in i ca l use, and a l so explain the potential hemolyzing e f f e c t on red c e l l s . There was grea te r oxidizing e f f e c t on glucose-6-phosphate dehydrogenase (G-6-PD) de f i c i en t than on normal erythrocytes. I t i s suggested t h a t adriamycin be used w i t h caution i n individuals w i t h G-6-PD def ic ien t red c e l l s .

Because adriamycin has

INTRODUCTION

Adriamycin (doxorubicin H C L ) has become one of the most f r e - quently used chemotherapeutic agents (1 ) . Unfortunately, i t s c l in i ca l use i s frequently associated w i t h ca rd io toxic i ty ( 2 ) . There is experimental evidence t o suggest t h a t this cardiac tox i -

735

Copyright 0 1980 by Marcel Dekker, Inc.

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736 SHINOHARA AND TANAKA

c i t y may be r e l a t e d t o g e n e r a t i o n o f r e a c t i v e oxygen compounds and l i p i d p e r o x i d a t i o n (3, 4 ) . Superoxide r a d i c a l i o n p r o d u c t i o n t h a t i s dependent on reduced n i co t i namide adenine d i n u c l e o t i d e phosphate has been demonstrated i n microsomes exposed t o an th ra - c y c l i n e s ( 3 ) . Adr iamycin has been shown t o induce severe c a r d i a c t o x i c i t y assoc ia ted w i t h p e r o x i d a t i o n o f c a r d i a c l i p i d s ( 4 ) .

Recently, Henderson and co l leagues have demonstrated t h a t ad r iamyc in generates oxygen r a d i c a l s i n r e d c e l l s (5 ) . pose o f t h i s s tudy was t o o b t a i n f u r t h e r knowledge o f t h e e f f e c t s o f ad r iamyc in on r e d c e l l s , and a l s o t o d i s c e r n i t s p o s s i b l e c l i n i - c a l s i g n i f i c a n c e .

The pu r -

MATERIALS AND METHODS

Commercial ly a v a i l a b l e ad r iamyc in ( d o x o r u b i c i n HC1) f rom A d r i a Labora to r ies , I nc . was d i s s o l v e d i n i s o t o n i c NaCl and added t o r e d c e l l suspensions i n v a r y i n g concen t ra t i ons .

The usual dosage o f adr iamycin, about 40 mg/M2, i s e q u i v a l e n t t o about 0.01 mg o f adriamycin/ml o f b lood. Genera l ly , concentra- t i o n s o f 0.01 mg/ml and 0.1 mg/ml were used f o r t h e in w b k o incuba- t i o n s t u d i e s ; concen t ra t i ons o f up t o 0.5 mg/ml were used f o r s t u d i e s o f t h e e f f e c t s o f ad r iamyc in on ATPase, c a t i o n f l u x , and osmot ic f r a g i l i t y .

Hepar in i zed b lood was washed 3 t imes w i t h i s o t o n i c NaCl and the b u f f y c o a t was removed a f t e r each c e n t r i f u g a t i o n . t h e in w b k c r i n c u b a t i o n s t u d i e s , r e d c e l l s were resuspended i n autologous plasma t h a t had been c e n t r i f u g e d a t h i g h speed (12000 g) t o remove leukocytes and p l a t e l e t s .

For t h e s tudy o f t h e e f f e c t s o f hemolysis d u r i n g t h e Selwyn- Dacie autohemolysis t e s t , ATPase, c a t i o n f l u x , and osmot ic f r a g i l i t y , d e f i b r i n a t e d b lood was used, because a t a c o n c e n t r a t i o n o f 0.5 mg a d r i amyci n/ml blood, hepar i n i zed b lood c l o t t e d .

Red c e l l g l y c o l y t i c pathway and hexose monophosphate shun t enzyme a c t i v i t i e s and reduced g l u t a t h i o n e (GSH) c o n t e n t were determined accord ing t o B e u t l e r ( 6 ) . measured by t h e method o f El lman eA d ( 7 ) . was assayed by t h e method o f Winterbourn eX UX (8) . v i t y was measured by t h e method o f Brewer eX d (9), and t h e a c t i - v i t y of Na-K-dependent ATPase was determined by t h e d i f f e r e n c e between t o t a l a c t i v i t y and t h a t i n t h e presence o f ouabain.

Red c e l l sodium and potass ium con ten t was determined by f l ame photometry. sured and t h e d i f f e rence determined f o r r e d c e l l content .

Fo r most of

A c e t y l c h o l i n e s t e r a s e was Superoxide dismutase

ATPase a c t i -

The contents o f whole b lood and o f plasma were mea-

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EFFECTS OF ADRIAMYCIN 737

Red c e l l membrane l i p i d p e r o x i d a t i o n was t e s t e d by t h e method o f Stocks and Dormandy ( l o ) , u s i n g t h e t h i o b a r b i t u r i c - a c i d (TBA) method f o r measuring ma lony ld ia ldehyde (MDA), a secondary p r o d u c t o f po lyunsa tu ra ted f a t t y a c i d pe rox ides .

et ul (11) . SDS po l yac ry lam ide g e l d i s c e l e c t r o p h o r e s i s o f t h e membrane p r o t e i n s was performed by t h e method o f Fai rbanks et ul i n 5% po lyac ry lam ide g e l (12) , and a l s o i n 3% ge l by t h e method o f Peacock and Dingman (13) by m i x i n g agarose t o l essen t h e con- c e n t r a t i o n o f po l yac ry lam ide . D i t h i o t h r e i t o1 was n o t used un less speci f i ca 11 y ment i oned .

Red c e l l membrane ghosts were prepared by t h e method o f Dodge

RESULTS

E f f e c t on hemolys is : Red c e l l s f rom d e f i b r i n a t e d b l o o d were i ncubated w i t h v a r y i n g concen t ra t i ons o f a d r i amyci n . D u r i n g i ncu- b a t i o n f o r 48 hours a t 37'C, 3.1% hemolys is was observed w i t h o u t adr iamycin, 3.2% w i t h 0.01 mg/ml adr iamycin, and 6.9% w i t h 0.1 mg/ml ad r iamyc in (mean o f 3 exper iments) i n each group. A d d i t i o n o f 10 mM g lucose i n t h e i n c u b a t i o n medium prevented hemolysis (< 2% a t a l l concen t ra t i ons o f ad r iamyc in ) .

v i t y . Both Na-K-ATPase and ouabain i n s e n s i t i v e ATPase were i n h i b i t e d a f t e r 2 hours o f i n c u b a t i o n a t ad r iamyc in concen t ra t i ons o f 0.1 mg/ml o r g r e a t e r ( F i g u r e 1 ) .

E f f e c t on r e d c e l l enzymes: Adr iamycin i n h i b i t e d ATPase a c t i -

The ATPase a c t i v i t i e s were n o t i n h i b i t e d

0001 41 02 05 ADRIAMYCIN (mq/ml)

FIGUIlE 1 Inhibition of AlI'Pase activity by a d r i a q c h . A----A Ouabain insensitive ATPase. activity w i t h aaaition of 20 p~ a++. of assays perfonred in dwlicate.

Na-K-ATPase, Closed symbols represent the

Lines represent mean d u e s

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738 SHINOHARA AND TANAKA

by adr iamycin concen t ra t i ons o f 0.2 mg/ml o r l e s s i f Cat+ was added i n a c o n c e n t r a t i o n o f 20 pM t o t h e i n c u b a t i o n m ix tu res . Red c e l l g l y c o l y t i c and hexose monophosphate shunt enzymes (G-6-PD and 6- phosphogl uconate dehydrogenase) as we1 1 as g l u t a t h i o n e reduc tase , g l u t a t h i o n e perox idase, ca ta lase , superox ide dismutase, and a c e t y l - cho l i nes te rase , were n o t i n h i b i t e d a f t e r 2 hours o f i n c u b a t i o n w i t h adr iamycin.

A f t e r 2 hours o f i n c u b a t i o n w i t h adr iamycin, r e d c e l l s ga ined sodium and l o s t potassium, a l t hough these e f f e c t s were observed o n l y a t a c o n c e n t r a t i o n o f ad r iamyc in o f 0.5 mg/ml b lood ( F i g u r e 2 ) . The osmot ic f r a g i l i t y o f these r e d c e l l s , however, was normal.

E f f e c t on g l u t a t h i o n e (GSH) s t a b i l i t y : GSH con ten t o f normal red c e l l s decreased on i n c u b a t i o n w i t h ad r iamyc in w i t h t i m e and i n c r e a s i n g adr iamycin concen t ra t i ons (F igu re 3 ) , w h i l e sulfhemo- g l o b i n c o n t e n t i nc reased (da ta n o t shown f o r su l fhemog lob in ) . Addi- t i o n o f glucose, adenosine, o r i nos ine , a t a c o n c e n t r a t i o n o f 10 mM, p r o t e c t e d GSH s t a b i l i t y . d e f i c i e n t r e d c e l l s even a t 0.01 mg/ml ad r iamyc in and was marked a t 0.1 mg/ml .

I n s t a b i l i t y o f GSH was apparent i n G-6-PD

E f f e c t on r e d c e l l GSH o f p a t i e n t s r e c e i v i n g adr iamycin: Venous b lood was ob ta ined 10 t o 15 minutes a f t e r p a t i e n t s had r e - c e i ved a d r i amyci n as an agent o f BACOP (B1 eomyci n , Adr i amyci n , Cycl o- phosphamide, Oncovin, and Prednisone) therapy.

lOfD G -100

5- -50

0321 0'1 0'2 05 ADRIAMYCIN (mq/ml)

Effect of adriartycin on cation flux. H Na o---o K Defibrinated blood was incubated w i t h adriamycin a t 40OC for 2 hours. Results of a single eqe rh t are shown, but multiple experiments danonstrated similar results.

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EFFECTS OF ADRIAMYCIN 739

0 5 to HOURS

FIGURE 3

Stability of reduced glutathione of noml control and G 6 - P D deficient red cells. cell samples incubated w i t h 0, 0.01, and 0.1 r q adrianycin/ml. 0, A, and 0 represent the values for 3 G-6-PD deficient red cell samples. - , ----, -.-. represent 0, 0.01, and 0.1 rrg adriamycin/ml red cell suspension, respectively, during incubation.

Vertical bars represent man ? SD for 6 llormal oontml red

These pa t i en t s were not G-6-PD def i c i en t . a t e l y a f t e r withdrawal was not low, nor d i d t he level of GSH decrease more than those of normal cont ro ls a f t e r incubation.

was slow, and when leve ls of malonyldialdehyde (MDA) generated were measured d i r e c t l y , the leve ls were low. Therefore, t he suscept i - b i l i t y of adriamycin incubated c e l l s t o hydrogen peroxide induced l i p i d peroxidation was investigated (Figure 4 ) . After incubation w i t h adriamycin f o r 24 hours, washed red c e l l s were exposed t o hydrogen peroxide along w i t h sodium az ide , a ca t a l a se i n h i b i t o r , and MDA formed was measured (10 ) . in te rac t ion of polyunsaturated l i p i d s and oxygen r ad ica l s , t h a t i s , peroxidation of l i p i d s . I t i s seen t h a t g rea t e r amounts of MDA formed with increasing adriamycin concentration.

amide gel e lec t rophores i s of red ce l l stroma (Figure 5a-c), incu-

GSH content immedi-

Effect on membrane l i p i d s : The process of l i p i d peroxidation

This test serves t o ind ica t e an

Effect on red ce l l membrane proteins: As shown by polyacryl-

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740 SHINOHARA AND TANAKA

0 1

T

FIGURE 4

E f f e c t of adrianych on mlonyldialdehyde (MDA) formtion. See text for detai ls . peroxide, and B to 10 mM hydmgm peroxide. mycin concentration as i n figure 3.

A represents the values when ~ e d to 7 nM hydrogen Each Line depicts adria-

bation of red c e l l s w i t h adriamycin f o r 24 hours resulted i n h i g h molecular weight complexes. Components 1 and 2 were primarily involved, t h a t i s , spec t r in , forming a la rge molecular weight complex (1 + 2 ) , which i s observed above spec t r in (bes t shown i n Figure 5c ) , and hemoglobin aggregated which remained on the top of the ge l , judged by location i n the gel according t o Palek et ul (14) . Also, band 3 may be involved, s ince the amount in t h e gel decreased with increasing concentrations o f adriamycin during incubation. de f i c i en t red c e l l s , and even a t 0.01 mg/ml, i t s e f f ec t was con- s iderable (Figure 5b). when the red c e l l s were incubated w i t h 10 mM glucose, o r when t he stromal protein was incubated with d i th io th re i t o l .

These e f f e c t s of adriamycin were marked i n G-6-PD

The amount o f aggregated proteins decreased

DISCUSSION

Red blood ce l l s contain superoxi de d i smutase which catalyzes the dismutation of superoxide rad ica ls t o oxygen and hydrogen per- oxide (15) ; the l a t t e r compound i s eliminated primarily by gluta- thione peroxidase (16) . Hemolytic compounds such as phenylhydra-

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EFFECTS OF ADRIAMYCIN 741

z i n e (17) and menadione (18) r e a c t w i t h hemoglobin t o produce s i g - n i f i c a n t amounts o f superox ide anion (02 ) . Recent s t u d i e s i n human e r y t h r o c y t e s i n d i c a t e t h a t ad r iamyc in i n t e r a c t s wi th oxyhemoglobin t o generate r e a c t i v e oxygen metabol ism ( 5 ) . Through t h e use of 1,4-naphthoquinone 2 -su l fona te , a s t r u c t u r a l analog o f menadione, i n r e d c e l l s dep le ted o f superox ide dismutase a c t i v i t y by an i n h i - b i t o r d i e t h y l d i t h i o c a r b a m a t e , Goldberg and S t e r n (19) have demon- s t r a t e d a t o x i c r o l e f o r 0 2 I n o u r in v&o experiments, ad r iamyc in ac ted indeed as a s low a c t i n g hemo ly t i c agent,although hemolys is occu r red o n l y a t h i g h concentra- t i o n s o f t h e d rug and was p reven tab le by t h e a d d i t i o n o f g lucose i n t h e i n c u b a t i o n m i x t u r e . p u r i f i e d superox ide dismutase may be i n a c t i v a t e d (20 ) . However, i n o u r s tud ies , t h e r e was no i n h i b i t o r y e f f e c t o f ad r iamyc in on r e d c e l l superox ide dismutase, ca ta lase , and on hexose monophosphate shunt and assoc ia ted enzymes g l ucose-6-phosphate dehydrogenase, 6-phosphogl uconi c dehydrogenase, g l u t a t h i o n e reductase, and g l u ta - t h i o n e perox idase. These r e s u l t s a r e c o n s i s t e n t w i t h an a c t i v e hexose monophosphate shunt when ad r iamyc in i s added t o human e ry - t h r o c y t e suspensions ( 5 ) .

Adr iamycin induces severe c a r d i a c t o x i c i t y assoc ia ted w i t h p e r o x i d a t i o n o f c a r d i a c l i p i d s i n m ice (4) . Our d a t a i n d i c a t e t h a t t h e r e a c t i v e oxygen m e t a b o l i t e s generated by ad r iamyc in i n r e d c e l l s cause g l u t a t h i o n e i n s t a b i l i t y and p e r o x i d a t i o n o f mem- brane l i p i d s . I n a d d i t i o n , t h e r e was o x i d a t i o n o f hemoglobin and membrane p r o t e i n s i n v o l v i n g components 1, 2, and 3, f o rm ing l a r g e mo lecu la r we igh t complexes when reduced g l u t a t h i o n e i s depleted.

Our data suppor t t h e r e c e n t f i n d i n g s o f Gosalvez and assoc ia tes (21) t h a t ad r iamyc in i s an i n h i b i t o r o f h e a r t microsomal Na-K-ATPase and t h a t ad r iamyc in a l s o i n h i b i t s potass ium t r a n s p o r t , b u t n o t sodium t r a n s p o r t , i n k idney s l i c e s . I n o u r s tudy o f r e d c e l l s , b o t h Na-K- ATPase and ouabain i n s e n s i t i v e ATPase were i n h i b i t e d , and potass ium was l o s t w i t h a g a i n i n sodium. Based on obse rva t i ons o f f r o g s k i n e p i t h e l i u m , S o l i e and Yuncker (22 ) b e l i e v e t h a t ad r iamyc in induces changes i n membrane p e r m e a b i l i t y t o sodium i o n s . Such e f f e c t s of ad r iamyc in on membrane ATPase and c a t i o n p e r m e a b i l i t y may e x p l a i n , i n p a r t , t h e c a r d i o t o x i c i t y o f t h i s drug. I t i s t o be no ted t h a t ad r iamyc in has a g l y c o s i d i c s t r u c t u r e which resembles some g l y c o - s i d e s w i th c a r d i o t o x i c e f f e c t . membrane p r o t e i n s and l i p i d s may have a r o l e i n t h e i n h i b i t i o n o f Na-K-ATPase. Indeed, ATPase i s known t o be l i p i d dependent. How- ever , another l i p i d dependent membrane enzyme, a c e t y l c h o l i n e s t e r a s e , was n o t i n h i b i t e d by adr iamycin. It i s o f i n t e r e s t t h a t t h e i n h i - b i t i o n o f Na-K-ATPase and e f f e c t on i o n t r a n s p o r t i s markedly reduced by Ca++, p robab ly by c h e l a t i o n o f t h i s meta l by ad r iamyc in (21) . Our r e s u l t s a r e c o n f i r m a t o r y o f t he s t u d i e s by Gosalvez and c o l - leagues i n t h i s regard.

i n c h e m i c a l l y induced hemolys is .

On pro longed exposure t o hydrogen pe rox ide ,

P e r o x i d a t i v e damage t o r e d c e l l

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742 SHINOHARA AND TANAKA

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EFFECTS OF ADRIAMYCIN 7 4 3

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744 SHINOHARA AND TANAKA

The concen t ra t i ons o f adr iamycin used i n uLtho t o t e s t i t s o x i d i z i n g hemo ly t i c e f f e c t s a r e cons ide rab ly h i g h e r than t h e r a - p e u t i c l e v e l s . of adr iamycin on r e d c e l l s a r e dependent on c o n c e n t r a t i o n and t ime o f i n c u b a t i o n , t he c l i n i c a l use o f ad r iamyc in would n o t be expected t o a f f e c t normal r e d c e l l s . However, t h e p o s s i b i l i t y t h a t adr iamycin a t h i g h dosage schedules may cause s u f f i c i e n t o x i d a t i v e i n j u r y t o G-6-PD d e f i c i e n t r e d c e l l s t o r e s u l t i n hemo- l y s i s should be considered. Another such c l i n i c a l s i t u a t i o n i s combinat ion chemotherapy i n v o l v i n g the use o f ad r iamyc in and BCNU (1, 3 -b i s (2-chloroethy1)-1-nitrosourea), which i n h i b i t s g l u t a - t h i o n e reductase (23) .

mycin have a l s o been observed i n r e d c e l l s . mycin on r e d c e l l s may serve as a model f o r f u r t h e r s tudy o f t h e mechanism o f c a r d i o t o x i c i t y t h a t occurs d u r i n g t h e c l i n i c a l use o f adr iamycin, and a l s o may account f o r t h e hemolysis observed i n o u r -in vL&u s t u d i e s .

A l though o u r i n u&o data show t h a t t h e e f f e c t s

Almost a l l o f t h e e f f e c t s on h e a r t t i s s u e a t t r i b u t e d t o a d r i a - These a c t i o n s of a d r i a -

ACKNOWLEDGEMENTS

Th is i n v e s t i g a t i o n was suppor ted by g r a n t AM 14898 f rom t h e Na t iona l I n s t i t u t e s o f Heal th . The au tho rs thank D r . N. Noble f o r rev iew ing the manuscr ip t .

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REFERENCES

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