Effect of Melatonin on Hemolysis Induced by Cyclosporine

대한신장학회지 : 제 20 권 제 5 호 2001년

1)

INT RO DUCT IO N

Hemoly sis is one of the side effect s of cyclo- sporine(CsA ) ^{1 - 4 )} , and the incidence during CsA

* T h e p r e s en t r e s e ar ch h a s b e en co n d u c ted b y th e Gr an t of K e im y u n g U n iv e r s it y , T aeg u , K or e a in 1999 . C o r r e s p o n d en ce t o : K y o - C h e ol M u n , M .D ., P h .D .

D ep a r tm e n t o f B ioc h em is t r y , K eim y u n g U n iv e r s ity S c h o o l o f M ed ic in e , 19 4 D o n g s a n - d o n g , J o o n g - g u , T ae g u , K o r e a

T e l : 82 - 53 - 25 0 - 7 786 , F a x : 8 2 - 5 3 - 252 - 16 05 E - m ail : m u n @d s m c .o r .k r

therapy is reported about t en percent ^{1 )} . Hemoly sis could indicate ischemic vascular damag e leading t o art eriolar medial hyalinosis preceding fibrosis in CsA nephr otoxicity ^{1 )} w hich is the m ost com - m on complication and clinically important side ef- fect of cyclosporine ^{5 - 8 )} . A report by Azuma, et al.

4 ) strongly sugg est ed that CsA - induced hemolysis is r esulted fr om the increased production of free r adical species by CsA . Melat onin, a pineal s ecre- t ory product , act s as a highly efficient free rad- ical scaveng er that protect s cells from the damag -

E f f e c t of M e la t on in on H e m oly s is In d u c e d b y C y c lo s p or in e

C h u n - S ik Kw a k , P h . D . , Ky o - C h e o l M u n , M . D . a n d S e o n g - Il S u h , M . D .

I nstitute f or M edical S cience, K eimy ung Univers ity S chool of M edicine, Taeg u, K orea

= A b s t r a c t =

H em oly sis is on e of th e side effect s of cyclosporin e (CsA ). S om e ex perim ent al an d clinical dat a h av e str ong ly sug g est ed th at Cs A - in duced hem oly sis is r esult ed fr om th e incr eas ed pr oduction of fr ee r adical species by CsA . M elat onin , a pin eal s ecr et ory pr oduct , act s as a hig hly efficient fr ee r adical scav eng er . T hu s , m elat onin m ay h av e a protectiv e effect on the CsA - induced hem olysis . T o test this hypothesis, the final con- centr ation of 4.2×10 ⁶ / m L w ith hum an ery thr ocyt es w as incub at ed in t est t ube at 37℃

w at er b ath w it h 1.67 m g/ m L of CsA an d 72 nm ol/ m L of m elat onin . T he deg r ee of h em oly sis an d th e am ount of m alon dialdehy de w hich giv es an in dir ect in dex of ox i- dativ e injury w er e m easur ed in g r oup 1 cont aining only is ot onic buffer s olution , in g r oup 2 cont aining only Cs A , an d in g r oup 3 cont aining b oth CsA an d m elat onin . T h e deg r ees of h em oly sis in gr oup 2 w er e hig h er th an th os e of g r oup 1. T h e deg r ees of hem oly sis in gr oup 3 w er e hig h er th an th os e of gr oup 1, an d low er th an th os e of group 2. T he amounts of malondialdehyde in group 2 w ere higher than thos e of gr oup 1.

T h e am ount s of m alon dialdehy de in g r oup 3 w er e hig h er th an th os e of gr oup 1, an d low er th an th os e of gr oup 2. T h es e r es ult s in dicat e th at th e dir ect cont act of erythr o- cyt es w ith CsA r esult s in fr ee r adical m ediat ed hem oly sis an d t he h em oly sis by Cs A can b e pr ev ent ed w ith m elat onin .

K e y W o r d s : Cy closporin e, H em oly s is , M alon dialdehy de, M elat onin , Ox y g en fr ee r adicals

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— Chun - Sik Kw ak , et al. : Effect of Melatonin on Hemoly sis Induced by Cyclosporine —

ing by oxyg en free r adicals 6 , 9 - 1 1 )

. T hus, m elat onin m ay hav e a prot ective effect on the CsA - induced hem olysis . T o t est this hypothesis , human eryth- r ocyt es w as incubat ed in t est tube at 37℃ w at er bath w ith CsA and melatonin . T he degree of he- molysis and the amount of malondialdehyde which giv es an indirect index of oxidative injury ^{12 )} w er e m easur ed.

S UBJ ECT S A ND MET HO DS 1 . E x p e rim e n t al P rot oc ol an d Grou p s

Human erythr ocytes w ere w ashed 5 times w ith phosphate buffered saline (PBS ) solution at pH 7.4 and t emperature of 4℃. Aft er w ashing , incubation w as performed on thr ee groups for 24, 48 and 72 hour s at 37℃ w at er bath . In group 1, the final concentration of 4.2×10 ⁶ / mL w ith hum an erythro- cytes w as incubat ed in the t est tube with PBS solution cont aining 0.17% ethanol. In group 2 con- t aining only CsA, the final concentration of 4.2×

10 ⁶ / mL w ith human erythr ocytes w as incubat ed in the test tube containing 1.67 mg of CsA/ mL of PBS s olution w ith 0.17% ethanol. In gr oup 3 containing CsA and m elat onin, the final concen- tration of 4.2×10 ⁶ / mL w ith hum an erythrocytes w as incubat ed in the test tube w ith 1.67 mg of CsA/ mL of PBS and 72 nm ol of m elatonin dis- s olved in 1 mL of 0.17% ethanol. T he experiment w er e done sev en tim e in each group.

2 . D etermin ati on of th e deg re e of h em oly s is

After incubation at 37℃ w ater bath, erythro- cytes w er e centrifuged at 2,000 rpm for 5 min . T he abs orbance of the supernat ant in each tube w as measures at 540 nm . T he extent of hemolysis w as calculated w ith standar d curv e obt ained from hem olysis w ith cold distilled w at er .

3 . D e t e rm i n ati on o f m al on di al d e h y d e

T he amount of malondialdehyde in the superna- t ant aft er centrifug ation w as measured by the thiobarbituric acid as say which is based on m al- ondialdehyde reacts w ith thiobarbituric acid t o giv e a r ed species absorbing at 535 nm ^{12 )} .

4 . S t at i s t ic al A n aly s i s

St atistical ev aluation of significant difference betw een means w as perform ed w ith the paired t - t est . p- v alues of < 0.05 w ere considered signifi- cant .

R ES ULT S

T he results are shown in T able 1. T he degrees of hem olysis in group 2 w ere significantly higher than those in group 1. Although the degrees of hem olysis in gr oup 3 w ere significantly higher than those in group 1, those in gr oup 3 w er e significantly low er than those in group 2.

T he lev els of m alondialdehyde in group 2 w er e

T ab le 1 . T he D e g re e of H e m oly s is and the A m oun t of M al ondi alde hy de

Incubation time

Gr oups

Deg ree of hem oly s is (% ) Am ount of MDA (nm ol/ mL ) Gr oup 1 Gr oup 2 Gr oup 3 Gr oup 1 Gr oup 2 Gr oup 3 24 hr s

48 hr s 72 hr s

3.2±0.5 43.6± 1.4^* 36.2± 2.5^{* ,§} 1.5±0.2 3.7± 1.2^* 1.9±0.4^{* , ‡} 5.7±0.5 67.5± 1.7^* 59.9±3.0^{* ,§} 2.0±0.2 9.4±0.3^* 5.3±0.8^{* ,§} 18.4± 1.1 79.5±4.4^* 67.9±4.6^{* , †} 3.3±0.2 12.2±3.2^* 7.3± 1.4^{* , †}

Values are m eans± S .D .,

p <0.001 vs . g roup 1,

p <0.05 vs . g roup 2,

p <0.01 vs. g roup 2 ,

p <0.001 vs. g roup 2 , Group 1 : control g roup , g roup 2 : CsA treate d g roup , g roup 3 : CsA and m e latonin treate d g roup , M D A : m alondialdehy de

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— 대한신장학회지 : 제 20 권 제 5 호 통권 제 71 호 2001 —

significantly higher than thos e in group 1. Al- though the am ount s of hem olysis in group 3 w er e significantly higher than those in group 1, those in gr oup 3 w er e significantly low er than those in gr oup 2.

D IS C US S IO N

Oxyg en free radicals can be defined as oxyg en m olecules or m olecular fr agm ent s that hav e an unpaired electr on ^{13 , 14 )} . T hey are form ed in all liv- ing org anism s during physiological and pathophy- siological m etabolism ^{13 , 1 4 )} , and cause cell and tis - sue damag es due to their high chemical r eactivity

1 3 , 14 )

. T hey can r eact w ith m acromolecules includ- ing lipid, prot ein and DNA ^{1 5 , 16 )} . P eroxidation of lipids expos ed to oxyg en fr ee radicals is responsi- ble for damag e to cells and tissues in viv o, where it may cause of CsA - induced toxicity including CsA nephrotoxicity 5 , 7 , 8 )

.

Am ong the s everal t oxicities by CsA , hem oly- sis is one of the side effects of cyclosporine ^{1 - 4 )} . CsA - induced hem olysis can be prev ent ed by vita- min E ^{4 )} w hich has antioxidant property .

Melatonin, a pineal s ecretory pr oduct, has been r eport ed to have antioxidant properties in addition t o it s know n hormonal activities 6 , 9 - 1 1 )

. It is an effectiv e scaveng er of both the highly toxic hy - droxyl radical and the per oxyl radicals 6 , 9 - 1 1 )

. Addi- tionally , it is r eported that melat onin m ay stim- ulate some important antioxidative enzym es such as superoxide dismut ase, glutathione peroxidase

6 ) . Like these, m elatonin act s as a highly efficient free radical scaveng er w hich prot ect s cells from the damaging by oxyg en free r adicals . T hus, mel- at onin might hav e a pr ot ectiv e effect on the CsA - induced hem olysis .

In this experiment , ther e w ere significant in- cr eases in levels of hemoly sis and malondialde- hyde in CsA - treat ed gr oup (group 2), suggesting a significant hem olysis due to oxyg en free radi- cals . Melat onin tr eatm ent in group 3 significantly

pr ot ected hem olysis and free radical mediat ed injury ag ainst CsA - induced chang es .

T hese result s indicate that the dir ect cont act of erythrocyt es with CsA result s in fr ee r adical m ediat ed hem olysis and the hem olysis by CsA can be partially prev ent ed w ith m elatonin .

= 국 문 요 약 =

Cy c l o s p o rin e 에 의 한 용 혈 에 서 M e l at o n in 의 효 과 계명대학교 의과대학 의과학연구소

곽춘식・문교철・서성일

장기 이식을 한 환자에서 빈혈이 합병증으로 동반되 는 것을 볼 수 있다 . 이러한 원인에 대하여는 cy- clospor ine (CsA )에 의한 용혈이 그 원인의 하나로 생각 되고 있으며 이 과정에 활성 산소가 매개하는 것으로 생각되고 있다 . 이 연구에서는 강력한 항산화제로 알려 진 melat onin에 의해 이러한 산화 작용이 방지되어 용 혈이 방지될 수 있는지를 관찰하고자 하였다 . 실험군을 첫째 제 1군인 대조군 , 둘째 제 2군인 CsA만 첨가한 군 , 셋째 제 3군인 CsA 및 melatonin을 첨가한 군으로 나누고 각 군의 용혈의 정도와 활성 산소에 의한 손상 정도를 측정하였다 . 용혈의 정도와 활성 산소에 의한 손상 지표인 malondialdehyde치는 모두 제 2군에서 제 1군에 비해 유의하게 증가하였으며, 제 3군은 제 1군 보다 유의하게 증가하나 제 2군 보다는 감소하였다 . 이 상의 결과로 보아 CsA는 적혈구에서 활성 산소로 인 한 손상을 증가시켜 용혈을 유발하는 것으로 생각되며 , 이러한 손상과 용혈은 항산화제인 melatonin에 의해 일부 방지 될 수 있을 것으로 생각된다 .

R E F E R E NC ES

1) N oel C, Saunier P , H azzan M , Pr uv ot F R , Dr a- con M , Jude B , Lelievre G : Incidence and clinic al pr ofile of m icr ov ascular com plication s in r en al al- log r afted patients tr eated w ith cyclospor ine . A nn

M e d I nterne 143 (Suppl 1) :33- 36, 1992

2) Uen o H , Ishiy am a T , Y am ada K , A kim ot o Y , Koike M , Nak am aki T , Hin o K, T om oy asu S , T suru oka N : Red cell fr ag ment ation syndr om e after bone m arr ow tr an splantation presum ed to be cau sed by cy clospor in A . R insho K ets uek i

34 :362- 366, 1993

3) Galli F C, Dam on LE , T om lan ovich SJ , Keith F , Ch atterjee K, DeM arco T : Cyclosporine - induced hem olytic ur emic sy ndr om e in a heart tr ans plant

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— 곽춘식 외 2인 : Cyclosporine에 의한 용혈에서 Melatonin의 효과 —

recipient . J H eart L ung T ransp lant 12 :440- 444, 1993

4) Azum a E , H ir ay am a M , Nak an o T , N ag ai M , Hir atake S , Kom ada Y , Sakur ai M : A cute he- m oly sis dur ing cyclosporine ther apy s ucces sfully tr eated w ith vitamin E . B one M arrow T ransp lant

16 :321- 322, 1995

5) al Kh ader A , al Sulaim an M , Kishore PN , M o- r ais C, T ariq M : Quinacrine attenu ates cyclospo- rine - induced nephr ot ox icity in r at s . T ransp lanta-

tion 6 2 :427- 435, 1996

6) Reiter RJ , Carneir o RC, Oh CS : Melat onin in re- lation t o cellular antiox idative defens e m echa- nism s . H orm M e tab R es 29 :363- 372, 1997 7) Zhong Z, Ar teel GE , Conn or HD, Yin M , F r an -

kenberg MV , Stachlew itz RF , R aleig h JA , M as on RP , T hur m an RG : Cyclosporin A increases hyp- ox ia and free r adical pr oduction in r at kidney s : prevention by diet ar y g lycine . A m

J P hy s iol 275 :F595- 604, 1998

8) Yang JJ , F inn WF : Effect of oxy pur in ol on cy - clospor ine toxicity in cultured EA , LLC- P K 1 and MDCK cells . R en F ail 20 :85- 101, 1998

9) Reiter RJ : Ox idative pr oces ses and antiox idativ e

defen se m ech anism s in the ag ing br ain . FA S E B

J 9 :526- 533, 1995

10) N ohl H , St aniek K : Is m elat onin an antiox idant ?

W ien K lin W ochens chr 109 :737- 740, 1997

m in E and melat onin as antiox idant s ag ain st lip- id per oxid ation in r at r etin al h om og enates . J P i-

neal R es 24 :239- 244, 1998

12) Bueg e JA , A u st SD : Micr os om al lipid per ox ida- tion . M ethods E nz y m ol 5 2 :302- 310, 1978 13) H og g N : F r ee r adicals in disease . S em in R ep rod

E ndocrinol 16 :241- 248, 1998

14) Gam aley IA , Klyubin IV : R oles of reactiv e oxy - g en species : s ign aling and r eg ulation of cellular function s . I nt R e v Cy tol 188 :203- 255, 1999 15) F antel A G, M ackler B, St am ps LD, T r an T T ,

Per s on RE : Reactive ox yg en species and DNA ox idation in fet al r at tis sues . F re e R adic B iol

M e d 25 :95- 103, 1998

16) Ch akr abar ti S K, B ai C, Subr am anian KS : DNA - pr otein cr os slink s induced by nickel com pounds in is olated r at r enal c ortical cells and its ant ag - onism by specific am in o acids and m ag nesium ion . T ox icol A pp l P harm acol 15 4 :245- 255, 1999

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