Dithranol, Glucose-6-phosphate Dehydrogenase Inhibition and Active Oxygen Species

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Sonderdruck I Reprint

Arzneimittel-Forschung/Drug Research

Arzneim.-Forsch./Drug Res. 41 (II), 11, 1176-1181 (1991) EDITIO CANTOR • D-7960 AULENDORF

Dithranol, Glucose-6-phosphate Dehydrogenase Inhibition and Active Oxygen Species

K . M ü l l e r , M . Seidel, C . B r a u n , K . Ziereis, a n d W. Wiegrebe

D e d i c a t e d to Professor D r . D r . Ernst M u t s c h i e r o n the occasion o f his 60th birthday

Summary

Inhibition of glucose-6-phosphate dehydrogenase (G6- PDH) by dithranpl (anthralin, CAS 480-22-8) has been studied in the presence of catalase, superoxide dismutase (SOD) and various scavengers of active oxygen species.

Most scavengers were found to be either inhibitors of G6- PDH by themselves or simply without effect. The com- bined addition of catalase and SOD as well as the heat- denatured enzymes and the oxygen radical scavengers a- tocopherol and salicylic acid markedly reduced the inhi- bitory effect of dithranol. The direct exposure of G6-PDH to active oxygen species led to different results. When lib- erated from a water-soluble naphthalene endoperoxide, singlet oxygen was without effect whereas photosensiti- zation with methylene blue resulted in a total loss of en- zyme activity. Experiments under anaerobic conditions revealed that this inhibition was accomplished by the tri- plet state of the sensitizer. Superoxide anion radical was highly effective at concentrations corresponding to the amount of that produced by a 10 nmol/l dithranol solu- tion. In contrast, hydroxyl, alkylperoxyl and alkoxyl rad- icals were all less efficient. H

2

0

2

and alkylhydroperoxides did not alter the enzyme activity. The results suggest that

Of is the potent species towards G6-PDH, if dithranol acts through formation of active oxygen species.

Zusammenfassung

Dithranol, Glucose-6-phosphat-Dehydrogenase-Hemmung und aktive Sauerstoffspezies

Glucose-6-phosphat-Dehydrogenase (Gö-PDH)-Hem- mung durch Dithranol (Anthralin, CAS 480-22-8) wurde in Gegenwart von Catalase, Superoxid-Dismutase (SOD) und verschiedenen Fängern für aktive Sauerstoffspezies untersucht. Die meisten Fänger waren entweder selbst Hemmstoffe der G6-PDH oder wirkungslos. Sowohl die kombinierte Zugabe von Catalase und SOD als auch die denaturierten Enzyme, ebenso die Sauerstoffradikalfän- ger a-Tocopherol und Salicylsäure zeigten eine deutliche Reduzierung der durch Dithranol verursachten Hemmefi fekte. Die direkte Einwirkung von aktiven Sauerstoffspe- zies auf G6-PDH führte zu unterschiedlichen Resultaten.

Freisetzung von Singulett-Sauerstoff aus einem wasser- löslichen Endoperoxid war ohne Effekt, hingegen führte die Photosensibilisierung mit Methylenblau zum vollstän- digen Verlust der Enzymaktivität. Experimente unter an- aeroben Bedingungen zeigten jedoch, daß diese Hem- mung durch den Triplett-Zustand des Sensibilisators ver- ursacht wurde. Superoxid-Anion-Radikal erwies sich in Konzentrationen, die von einer 10 jumol/l Dithranol-Lö- sung produziert werden, als hochwirksam. Hydroxyl-, Alkylperoxyl- und Alkoxyl-Radikale waren hingegen we-

niger wirksam. H

²

0

²

und Alkylhydroperoxide führten zu keiner Beeinträchtigung der Enzymaktivität. Die Resul- tate deuten darauf hin, daß Oy die potente Spezies der G6-PDH-Hemmung durch Dithranol ist, wenn diese über die aktiven Sauerstoffspezies erfolgt.

Key words: Anthralin, in vitro studies • Antipsoriatic drugs • CAS 480-22-8 • Dithranol • Free radicals • Glucose-6-phosphate dehydrogenase, inhibition

1. Introduction Hl- ^

ne m

° d e o f action o f the drug at the molecular level is still not k n o w n i n detail, but it may act, at least i n part, D i t h r a n o l (anthralin, l a ; C A S 480-22-8) has been used as o n m a i n l y four cellular targets: a) interaction w i t h D N A an effective topical antipsoriatic drug for over 70 years [2—6], b) alteration o f m i t o c h o n d r i a l functions [7—10],

c) i n h i b i t i o n o f the 5- a n d 12-lipoxygenase pathways o f

arachidonic a c i d m e t a b o l i s m [11 — 13], a n d d) i n h i b i t o r y

action o n cytosolic enzymes o f the glycolytic pathway,

such as glucose-6-phosphate dehydrogenase ( G 6 - P D H )

Institute of Pharmacy, University of Regensburg [14—18]. T h i s key enzyme o f the hexose monophosphate

(Fed. Rep. of Germany) shunt is elevated i n psoriatic skin [19, 20]. In view o f the

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instability o f d i t h r a n o l i n weakly basic aqueous solution [18], it is u n l i k e l y that the molecule itself is responsible for the b i o l o g i c a l properties. Consequently, the i n h i b i - tory effects might be related to d e c o m p o s i t i o n products formed d u r i n g the a u t o x i d a t i o n o f d i t h r a n o l [18]. Since the o n l y two k n o w n o x i d a t i o n products, the d i m e r b i a n - throne a n d the anthraquinone d a n t h r o n (3), are o n l y moderate i n h i b i t o r s o f G 6 - P D H , it has been suggested that other b r e a k d o w n products must be the most active species towards G 6 - P D H [18].

T h e free r a d i c a l m e c h a n i s m o f d i t h r a n o l o x i d a t i o n starts w i t h d e p r o t o n a t i o n followed by electron abstraction to give l,8-dihydroxy-9-anthrone-10-yl-radical (2) [21, 22].

Because o f its stability a n d inertness towards oxygen this r a d i c a l was d i s c o u n t e d as an i m p o r t a n t intermediate [23]. In the presence o f oxygen, the univalent reduction o f oxygen by d i t h r a n o l a n i o n results i n the f o r m a t i o n o f superoxide a n i o n ('0

2

") [24, 25] (Scheme 1 (1)), d i s m u - tation o f this species generates hydrogen peroxide [24], a n d by the iron-catalyzed Haber-Weiss-reaction the far more reactive h y d r o x y l radical ( O H ) is p r o d u c e d [26].

P h o t o c h e m i c a l l y , electronically excited m o l e c u l a r oxy- gen (singlet oxygen,

!

0

2

) is p r o d u c e d by the d i t h r a n o l a n i o n ( l b ) [27, 28], w h i c h i n t u r n is o x i d i z e d to d a n t h r o n

(3, Scheme 1 (2)) i n a self-sensitized process most prob-

ably v i a an endoperoxide [27].

OH O OH

3 Scheme 1

A l t h o u g h this p r o d u c t i o n o f active oxygen species i n v i t r o by d i t h r a n o l is well documented, it has not yet been es- tablished whether this property is relevant to the mech- a n i s m o f action o f a n t h r a l i n o n b i o l o g i c a l targets. D i t h - r a n o l interacts irreversibly w i t h G 6 - P D H [18]. Irrevers- ible i n h i b i t i o n may be d e r i v e d from an u n d e t e r m i n e d re- dox pathway i n v o l v i n g oxygen radicals generated by d i t h r a n o l , resulting i n an inactive enzyme through de- struction o f specific a m i n o acids, p a r t i c u l a r l y h i s t i d i n e a n d t r y p t o p h a n [29]. Studies not related to d i t h r a n o l showed that the m e m b r a n e - b o u n d S H enzyme glyceral- dehyde-3-phosphate dehydrogenase was i n h i b i t e d by ox- i d a t i o n o f the S H groups by active oxygen species [30].

T h e objective o f the present study was to assess the role o f active oxygen species i n the i n h i b i t o r y action o n G 6 - P D H by d i t h r a n o l . T h i s i n h i b i t i o n is u t i l i z e d for a first screening for antipsoriatic a c t i v i t y to date [31, 32]. D i s - regarding the question whether G 6 - P D H i n h i b i t i o n is a significant m e c h a n i s m for the mode o f action or not we f i n d this test a useful t o o l i n our efforts to o b t a i n more insight into the nature o f the i n h i b i t o r y species. I f d i t h -

ranol acts through active oxygen f o r m a t i o n , a d d i t i o n o f catalase, superoxide dismutase ( S O D ) , a n d various oxy- gen radical scavengers to the reaction system should can- cel or d i m i n i s h the rate o f i n a c t i v a t i o n o f G 6 - P D H by d i t h r a n o l . A n o t h e r approach w h i c h w o u l d allow us to un- derstand the influence o f oxygen derivatives on this re- action system is to test the effects o f active oxygen species

— generated independently o f d i t h r a n o l — o n G 6 - P D H activity.

Abbreviations

B A S — b o v i n e serum a l b u m i n

D A B C O - l,4-diazabicyclo[2.2.2] octane D T P A — diethylenetriaminepentaacetic a c i d E D T A — ethylenediaminetetraacetic a c i d G 6 - P — glucose-6-phosphate d i s o d i u m salt

G 6 - P D H — glucose-6-phosphate dehydrogenase ( E C . 1.1.1.49) H S A — h u m a n serum a l b u m i n

N A D P + — n i c o t i n a m i d e adenine d i n u c l e o t i d e phosphate diso- d i u m salt

N B T — n i t r o blue t e t r a z o l i u m

N D P 02 — endoperoxide o f 3,3,-(l,4-naphthylene)dipropionate N D G A — n o r d i h y d r o g u a i a r e t i c a c i d

S O D — superoxide dismutase ( E C . 1.15.1.1) X O — xanthine oxidase ( E C . 1.1.3.22)

2. Materials and methods 2.1. Chemicals

D i t h r a n o l (anthralin, l,8-dihydroxy-9(10H)-anthracenone) was prepared by r e d u c t i o n o f d a n t h r o n [33] a n d p u r i f i e d by c o l u m n chromatography ( S i 0²/ C H²C 1²) ; N D P 0² was prepared accord- ing to the m e t h o d o f X u b r y [34]; B S A , ß - c a r o t e n e , catalase from b o v i n e l i v e r ( E . C 1.11.1.6; 20000 U / m g protein), chelating resin ( s o d i u m form), deferoxamine mesylate, 2 , 2 ' - d i p y r i d y l , D T P A , G 6 - P , glutathione, H S A , N A D P ^ , N D G A , p r o p y l gallate, pyrogallol, S O D from b o v i n e erythrocytes (2800 U / m g protein), x a n - thine, X O (Sigma, M u n i c h , F R G ) , cumene hydroperoxide, E D T A , F e C l3 • 6 H20 , F e S 04 • H20 , hydrogen peroxide (30 %), m a n n i t o l , methylene blue, N B T , s o d i u m citrate, a-tocopherol ( E . M e r c k , D a r m s t a d t , F R G ) , ß - a l a n i n e , ascorbic a c i d , L-cysteine, D A B C O , glycine, rose bengal, salicylic a c i d , L-serine, s o d i u m benzoate, thiourea ( A l d r i c h , S t e i n h e i m , F R G ) . G 6 - P D H (350 U / m g p r o t e i n ; Boehringer M a n n h e i m G m b H , M a n n h e i m , F R G ) , tert-butyl h y d r o p e r o x i d e (Janssen, N e t t e d a l , F R G ) .

2.2. G6-PDH assay

I n c u b a t i o n experiments were performed i n a R i n g e r buffer c o m - posed o f 140 mmol/1 N a C l , 2.7 mmol/1 K C l , 5 mmol/1 N a H C 0³,

1.8 mmol/1 C a C l2 a n d 1.1 mmol/1 M g C l2 at p H 7.5. T h e c o m - m e r c i a l enzyme suspension (350 U / m g ) was d i l u t e d 1:2000 i n the buffer a n d kept i n an ice bath (at t i m e zero the c o n t r o l ac- t i v i t y was 70 U / l ) . D i t h r a n o l stock solutions (4.4 mmol/1 i n acetone) were kept i n the dark under N2. In t y p i c a l runs G 6 - P D H a c t i v i t y was d e t e r m i n e d i n four different i n c u b a t i o n sets (final v o l u m e - 5 m l ) consisting o f (a) 4.48 m l o f R i n g e r buffer, 0.5 m l o f enzyme s o l u t i o n a n d 0.02 m l acetone, (b) 4.48 m l o f R i n g e r buffer, 0.5 m l o f enzyme s o l u t i o n a n d 0.02 m l o f d i t h r a n o l stock s o l u t i o n , (c) 3.98 m l o f R i n g e r buffer, 0.5 m l o f enzyme solution, 0.5 m l o f oxygen scavenger s o l u t i o n a n d 0.02 m l acetone, and (d) 3.98 m l o f R i n g e r buffer, 0.5 m l o f enzyme s o l u t i o n , 0.5 m l o f oxygen scavenger s o l u t i o n a n d 0.02 m l o f d i t h r a n o l stock so- l u t i o n . Test solutions were incubated for 1 h at 37 °C i n a shaking thermostat bath i n the dark. After i n c u b a t i o n , 0.5 m l o f the test s o l u t i o n was pipetted i n a 3.0 m l cell, 2.4 m l o f R i n g e r buffer a n d 0.05 m l o f a 0.03 mmol/1 N A D P⁺ solution were added, m i x e d a n d kept for 5 m i n at 25 °C. After a d d i t i o n o f 0.05 m l o f a 0.04 mmol/1 G 6 - P s o l u t i o n the rate o f increase i n absorbance at 339 n m was measured over a p e r i o d o f 5 m i n o n a U v i k o n 810 spectrophotometer ( K o n t r o n Instruments, E c h i n g , F R G ) .

2.3. Catalase assay

Catalase a c t i v i t y was measured f o l l o w i n g the d e c o m p o s i t i o n o f H²0² at 240 n m [35]. R e a c t i o n mixtures contained 1.98 m l o f phosphate buffer, 1 m l o f 0.03 mmol/1 H202 a n d 0.02 m l o f en- z y m e d i l u t i o n (2 U / m g protein). I n c u b a t i o n experiments for the d e t e r m i n a t i o n o f catalase i n h i b i t i o n by d i t h r a n o l were per- f o r m e d for 30 m i n at 37 °C i n a shaking thermostat bath under light p r o t e c t i o n . C o n t r o l : acetone.

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2.4. SOD assay

S O D a c t i v i t y was d e t e r m i n e d by measuring the i n h i b i t i o n o f pyrogallol a u t o x i d a t i o n , m o n i t o r e d s p e c t r o p h o t o m e t r i c a l l y at 420 n m [36]. I n c u b a t i o n experiments for the d e t e r m i n a t i o n o f S O D i n h i b i t i o n by d i t h r a n o l were performed for 60 m i n at 37 °C i n a shaking thermostat bath i n the dark. C o n t r o l : acetone.

2.5. Active oxygen species generating systems

A l l i n c u b a t i o n experiments for G 6 - P D H i n h i b i t i o n by active oxygen species were performed aerobically i n a phosphate buffer c o m p o s e d o f 50 mmol/1 K²H P 0⁴ • 3 H²0 a n d 5 mmol/1 K H²P 0⁴ at p H 7.8 under shaking. I n c u b a t i o n mixtures c o n t a i n e d 4.47 or 4.48 m l o f phosphate buffer, 0.5 m l o f enzyme s o l u t i o n a n d 0.02 or 0.03 m l o f an active oxygen generating system. I n c u b a t i o n t i m e was 30 m i n .

' 0² was p r o d u c e d either by means o f a dye-sensitized photo- c h e m i c a l m e t h o d or w i t h a c h e m i c a l source. In the case o f pho- tosensitized o x i d a t i o n s the i n c u b a t i o n mixtures were i r r a d i a t e d w i t h a c o o l e d halogen l a m p ( O s r a m Halostar, 100 W ) i n the presence o f rose bengal or methylene blue ( 1 0⁵ mmol/1), respectively.

A 1 % s o l u t i o n o f K²C r²0⁷ i n H²0 was used as a cutoff filter (550 nm). C o n t r o l s : A r / i n the dark. A s a c h e m i c a l source the d e c o m - p o s i t i o n o f the water-soluble N D P 02 [34, 37] (Scheme 2) was used.

COONa 37°C

COONa

COONa '02

NDP0² Scheme 2

T h e d e c o m p o s i t i o n o f the e n d o p e r o x i d e was e x a m i n e d at 37 °C i n the phosphate buffer described above. T h e disappearance o f N D P 0² (10 umol/1 i n the i n c u b a t i o n m i x t u r e o f 5 m l ) was m o n - i t o r e d by H P L C ( K o n t r o n 420, c o l u m n : N u c l e o s i l - 1 0 0 R P 18;

methanol/water/acetic a c i d (77:23:0.1); U V detection at 232 n m , K o n t r o n U v i k o n 735 L C ) . T h e first order rate constant was 4.2

± 0.2 • 1 0⁴ s"¹ w h i c h corresponds to a half-life o f 28 m i n . In order to produce a flux o f 02" a system o f X O a n d xanthine was used [38, 39]. G e n e r a t i o n o f 0² was ascertained spectro- p h o t o m e t r i c a l l y by m o n i t o r i n g the r e d u c t i o n o f N B T at 560 n m [40]. T h e amounts o f X O (0.02 U / m l ) a n d xanthine (30 umol/1) used were chosen o n the basis o f an I C^{5 0} o f d i t h r a n o l for G 6 - P D H o f about 10 umol/1. T h u s , the system was adjusted i n such a m a n n e r that the r e d u c t i o n rate o f N B T corresponded to 55 % [24] o f that caused by a 10 umol/1 d i t h r a n o l s o l u t i o n (45 % o f N B T r e d u c t i o n are due to direct electron transfer from d i t h r a n o l to N B T , for details o n 02" p r o d u c t i o n d u r i n g d i t h r a n o l a u t o x i - d a t i o n see [24]). X O was a d d e d last to initiate the reaction. T h e phosphate buffer s o l u t i o n was passed through a c o l u m n o f chelating resin to remove traces o f i r o n [41]. D T P A [42, 43] (0.1 mmol/1) or deferoxamine mesylate [44] (0.1 mmol/1) was a d d e d to prevent O H f o r m a t i o n .

O H was generated f r o m the x a n t h i n e / X O system either by ad- d i t i o n o f F e3 + - E D T A [42, 45] ( i r o n - c a t a l y z e d Haber-Weiss reaction) or F e²+ - D T P A [46] (superoxide d r i v e n F e n t o n reaction).

T h e final concentrations i n the reaction m i x t u r e s were 0.1 m m o l / 1 for F e C l3 • 6 H20 or F e S 04 • H20 , respectively. C o n t r o l s were p e r f o r m e d w i t h the ferrous salt, H²0², xanthine, and the chela- tors alone. A d d i t i o n a l l y , h y d r o x y l radicals were produced i n a F e n t o n reaction w i t h hydrogen peroxide a n d F e2 +- D T P A [46].

H²0² was a d d e d last i n three p o r t i o n s (final concentration 0.18 mmol/1) to initiate the reaction.

A l k o x y l radicals were generated by the catalytic d e c o m p o s i t i o n o f tert-butyl h y d r o p e r o x i d e [47] a n d cumene h y d r o p e r o x i d e by F e^{2 +} - d i p y r i d y l [48], whereas F e^{3 +} as a catalyst was e m p l o y e d to p r e d o m i n a n t l y generate a l k y l p e r o x y l radicals [49, 50]. Incuba- t i o n experiments c o n t a i n e d 0.1 mmol/1 o f the a l k y l h y d r o p e r o x - ide, 0.1 mmol/1 ferrous d i p y r i d y l or i r o n D T P A , respectively.

3. Results and discussion 3.1. Effects of catalase and SOD on the system G6-PDH/dithranol

Since d i t h r a n o l was reported to l o w e r the activities o f b o t h the enzymes [51], we e x a m i n e d the activities o f catalase a n d S O D as a function o f d i t h r a n o l c o n c e n t r a t i o n . The I C^{5 0} o f d i t h r a n o l for catalase was found to be 5 u m o l / 1 ( F i g . 1). W i t h a d i t h r a n o l c o n c e n t r a t i o n o f 1.7 • 1 05

mol/1 catalase activity decreased to about 70 % o f i n i t i a l a c t i v i t y after 30 m i n o f i n c u b a t i o n ( F i g . 2). A l t h o u g h this i n h i b i t i o n o f catalase by d i t h r a n o l has to be considered, there s t i l l r e m a i n s sufficient a c t i v i t y for studying the effect o f catalase o n G 6 - P D H i n h i b i t i o n by d i t h r a n o l under these c o n d i t i o n s .

C o n t r a r y to catalase the m a x i m a l degree o f i n h i b i t i o n o f S O D that c o u l d be a c h i e v e d was 35 % ( F i g . 3), even at higher concentrations o f d i t h r a n o l .

.1 100-

hibil

80-

a

60- 40- 20"

o -

.1 1 10 100 [uM]

Fig. 1: Inhibition of catalase as a function of dithranol concentration (ab- scissa). Incubation mixtures contained catalase (5 ug/ml) and indicated concentrations of dithranol in a final volume of 1 ml phosphate buffer for 30 min at 37 °C in a shaking water bath. Results are the average of 3 ex- periments (SD < 5 %).

Fig. 2: Time course of inhibition of catalase by dithranol. Incubation mixtures contained catalase (5 ug/ml) and 1.7 x 105 mol/1 dithranol in a final volume of 1 ml phosphate buffer for 30 min at 37 °C. Results are the average of 3 experiments (SD < 5 %).

S 40n

•a

0

'£

1

^30.

20-

10

0 20 40 60 80

[»iM]

Fig. 3: Inhibition of S O D as a function of dithranol concentration (ab- scissa). Incubation mixtures contained S O D (10 ug/ml) and indicated con- centrations of dithranol in a final volume of 2 ml phosphate buffer for 1 h at 37 °C in a shaking water bath. Results are the average of 3 experiments (SD < 5 %).

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D a t a from Table 1 reveal that neither catalase nor S O D show significant changes i n d i t h r a n o l efficacy towards G 6 - P D H w h e n added to the i n c u b a t i o n sets. O n the other hand, c o m b i n e d treatment w i t h catalase a n d S O D at high concentrations m a r k e d l y lowered i n h i b i t o r y action o n G 6 - P D H by d i t h r a n o l . T h e c o m b i n e d a d d i t i o n o f the en- zymes to the system removes b o t h H

²

0

²

(catalase) a n d

0

2

" ( S O D ) , a n d — by interaction o f these two species — O H as a secondary reaction product, as previously shown for d i t h r a n o l [26]. H o w e v e r , the denatured en- zymes were effective, too. M o r e o v e r , a d d i t i o n o f the pro- teins B S A or H S A led to a total loss o f i n h i b i t o r y action.

S i m i l a r although weaker effects were obtained w i t h a m i n o acids.

These results allow two possible explanations for the de- crease o f G 6 - P D H i n h i b i t i o n by d i t h r a n o l . F i r s t l y , they suggest an interaction o f d i t h r a n o l w i t h proteins or a m i n o acids. Observations f r o m other studies that d i t h - ranol is partially b o u n d or inactivated by serum proteins [17, 52—54] are i n agreement w i t h this interpretation.

Secondly, it is k n o w n that even catalase a n d S O D pro-

Table 1: Effects of catalase, S O D , proteins and amino acids on G 6 - P D H inhibition by dithranol.

Scavenger added % Inhibition

None 6 8 ± 4a>

Catalase (2 U/ml) 66 + 2

Catalase (300 U/ml) 52 + 4

Superoxide dismutase (28 U/ml) 6 5 ± 6

Superoxide dismutase (175 U/ml) 5 5 ± 2

Catalase (300 U/ml) + superoxide dismutase (175 U/ml) 1 4 ± 1 Heated catalase (300 U/ml) + heated superoxide

dismutase (175 U/ml) 1 7 ± 3

BSA (30 ug/ml) 0

H S A (30 jig/ml) 0

ß - A l a n i n e ( 1 . 6 x 1 04 mol/1) L-Cysteine (1.6 x 10"4 mol/1) Glutathione (1.6 x 1 04 mol/1)

6 0 ± 3 ß - A l a n i n e ( 1 . 6 x 1 04 mol/1)

L-Cysteine (1.6 x 10"4 mol/1) Glutathione (1.6 x 1 04 mol/1)

31 ± 4 ß - A l a n i n e ( 1 . 6 x 1 04 mol/1)

L-Cysteine (1.6 x 10"4 mol/1)

Glutathione (1.6 x 1 04 mol/1) 4 4 ± 5

Glycine (1.6 x 1 04 mol/1) 5 0 ± 1

L-Serine(1.6 x 1 04 mol/1) 3 8 ± 5

Incubation mixtures contained 7 m U / m l G 6 - P D H , 1.7 x 1 05 mol/1 dith- ranol and indicated concentrations of scavengers at 37 °C in Ringer buffer (30 min). % Inhibition is expressed as mean ± SD; n > 3. All scavengers added did not impair the enzyme activity (controls without dithranol). a )

n = 22.

teins (e.g. the inactive enzymes) react w i t h active oxygen derivatives [55]. T h e same is true for other proteins i n - c l u d i n g heat-denatured S O D a n d catalase [55]. A m i n o acids are highly reactive w i t h O H radicals [56]. T h u s , scavenging o f active oxygen species p r o d u c e d by d i t h - ranol may be responsible for the observed decline o f G D 6 - P D H i n h i b i t i o n i n the presence o f catalase, S O D a n d proteins.

Table 2: Effects of singlet oxygen quenchers and oxygen radical scavengers on G 6 - P D H inhibition by dithranol.

Scavenger added % Inhibition without

dithranol % Inhibition

None 0 6 8 ± 4a )

Ascorbic acid (1.6 x 1 04 mol/1) 28 ± 1 8 4 ± 4 ß-Carotene (8 x 106 mol/1) 4 4 ± 6 81 ± 5

D A B C O (1.6 x 10-3 mol/1) 0 9 3 ± 1

Mannitol (1.6 x 1 03 mol/1) 0 7 8 ± 3

N D G A (1.6 x 10-5 mol/1) 100 100

Propyl gallate (1.6 x 1 06 mol/1) 100 100 Pyrogallol (1.6 x 1 06 mol/1)

Salicylic acid (1.6 x 10*4 mol/1) Sodium benzoate (1.6 x 1 03 mol/1)

100 100

Pyrogallol (1.6 x 1 06 mol/1) Salicylic acid (1.6 x 10*4 mol/1) Sodium benzoate (1.6 x 1 03 mol/1)

0 5 6 ± 1

Pyrogallol (1.6 x 1 06 mol/1) Salicylic acid (1.6 x 10*4 mol/1)

Sodium benzoate (1.6 x 1 03 mol/1) 0 7 6 ± 2 Sodium citrate (1.6 x 1 04 mol/1) 0 7 2 ± 3

Thiourea (1.6 x 1 03 mol/1) 0 84 + 2

a-Tocopherol (3.2 x 105 mol/1) 2 9 ± 6 5 0 ± 6 Incubation mixtures contained 7 m U / m l G 6 - P D H , 1.7 • 1 05 mol/1 dith- ranol and indicated concentrations of scavengers at 37 °C in Ringer buffer (30 min). % Inhibition is expressed as mean ± SD; n > 3 .a ) n = 22.

O u r results demonstrate that most o f the oxygen radical scavengers were G 6 - P D H i n h i b i t o r s by themselves at concentrations necessary for the i n a c t i v a t i o n o f oxygen free radicals p r o d u c e d by d i t h r a n o l . T h i s indicates that experiments w i t h the system G 6 - P D H / d i t h r a n o l / s c a v - enger are not m u c h o f an appropriate tool i n search o f the active intermediate towards G 6 - P D H .

3.3. Effects of active oxygen species on G6-PDH activity

If the toxic effects o f d i t h r a n o l o n G 6 - P D H occurred by oxygen free radicals or singlet oxygen, a direct exposure o f the enzyme to these species s h o u l d lead to loss o f ac- tivity. Since d i t h r a n o l i n h i b i t e d this enzyme at an I C

^{5 0}

value o f about 10 umol/1 ( F i g . 4), the effects o f active oxygen species o n G 6 - P D H activity were tested at con- centrations corresponding a p p r o x i m a t e l y to the amount o f active oxygen species generated by a 10 umol/1 d i t h - ranol s o l u t i o n .

3.3.1. Singlet oxygen

' 0

²

causes damage to a variety o f biological targets [59].

D e s t r u c t i o n o f key active-site a m i n o acids leads to inac- t i v a t i o n o f m a n y enzymes. Indeed, studies o n G 6 - P D H p h o t o o x i d a t i o n by rose bengal resulted i n a specific de- struction o f h i s t i d i n e residues [60]. In this study we made use o f two different

¹

0

²

- s o u r c e s . P h o t o s e n s i t i z a t i o n w i t h rose bengal leading to a total loss o f a c t i v i t y (Table 3, entry 1) was consistent w i t h a previous report [60]. H o w - ever, pertinent experiments i n the dark revealed that even ground-state rose bengal is an i n h i b i t o r o f G 6 - P D H . C o n t r a r y , the ^ - s e n s i t i z e r methylene blue d i d not i n - h i b i t the enzyme i n the dark. A l t h o u g h photosensitiza-

3.2. Effects of singlet oxygen quenchers and oxygen radical scavengers on the system G6-PDH/dithranol

W i t h the exception o f the singlet oxygen quenchers ß- carotene [57] a n d ascorbic a c i d [58], scavengers o f oxy- gen radicals, such as m a n n i t o l , s o d i u m benzoate, s o d i u m citrate, salicylic a c i d , a n d thiourea d i d not influence G 6 - P D H a c t i v i t y o f controls (no d i t h r a n o l ) to an appreciable a m o u n t (Table 2). H i g h e r concentrations o f scavengers d i d i n h i b i t G 6 - P D H activity. In a d d i t i o n , the free r a d i c a l scavengers N D G A , p r o p y l gallate a n d pyrogallol are stronger inactivators o f G 6 - P D H than d i t h r a n o l . P e r t i - nent experiments revealed that a d d i t i o n o f o n l y s m a l l amounts o f these scavengers resulted i n a total loss o f enzyme activity. O f all tested c o m p o u n d s o n l y a-tocoph- erol, although an i n h i b i t o r by itself, a n d salicylic a c i d d i d weaken the i n h i b i t o r y effect o f d i t h r a n o l .

O H ' ' — 1 ' —

1 10 100 [uM]

Fig. 4: Inhibition of G 6 - P D H as a function of dithranol concentration (ab- scissa). Incubation mixtures contained 7 m U / m l G 6 - P D H and indicated concentrations of dithranol in a final volume of 5 ml phosphate buffer at 37 °C in a shaking water bath (30 min). Results are the average of three experiments (SD < 5 %).

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Table 3: Effects of active oxygen species on G 6 - P D H .

Entry Active oxygen species-generating

system % Inhibition

1 2 3 4 5 6

•o

² rose bengal (10 umol/l)/O2/100 W rose bengal (10 umol/l)/darka )

methylene blue (10 umol/l)/O2/100 W methylene blue (10 umol/l)/Ar/100 Wa>

methylene blue (10 (imol/l)/darka )

N D P 02 (10 umol/1)

100 100 100 100 0 0 7

8 9

o

²

-

xanthine (30 umol/l)/XO (0.02 U / m l / D T P A (100 mmol/1)

xanthine (30 umol/l)/XO (0.02 U/ml)/

E D T A ( 1 0 0 umol/1)

xanthine (30 umol/l)/XO (0.02 U/ml)/

deferoxamine (100 umol/1)

6 0 ± 3 6 2 ± 5 5 9 ± 4 10

11

02/ O H xanthine (30 umol/l)/XO (0.02 U/ml)/

D T P A (100 umol/l)/Fe2+ (100 umol/1) xanthine (30 umol/l)/XO (0.02 U/ml)/

E D T A (100 umol/l)/Fe3 + (100 umol/1)

5 9 ± 4 5 9 ± 4 12

13 14

' O H F e S 04 (100 u m o l / l ) / H202 (180 umol/1)/

D T P A (100 umol/1) FeSO4(100 umol/l)a )

F e S 04 (100 umol/l)/DTPA (100 umol/

l)a )

1 6 ± 1 1 7 ± 1 2 ± 0

15 H202 H2O2( 1 0 0 umol/1) 0

16 17

R O O H tert-butyl hydroperoxide (100 umol/1) cumene hydroperoxide (100 umol/1)

0 0 18

19

R O ' tert-butyl hydroperoxide (100 umol/1)/

dipyridyl-Fe2 + (100 umol/1)

cumene hydroperoxide (100 umol/l)/di- pyridyl-Fe2 + (100 umol/1)

1 4 ± 1 1 2 ± 1 20 R O O ' cumene hydroperoxide (100 umol/1)/

^{2 +}

to finally generate O H (Schemes 3 - 5 ) [63].

A d d i t i o n o f the chelator D T P A almost completely can- celled this i n h i b i t i o n , since ferrous D T P A is relatively stable to o x i d a t i o n [63]. T h e effects o f alkylperoxyl a n d

2 F e2 + + 202 *~ 2 F e3- + 2 -02"

Scheme 3

2 -0²- + 2H⁺ *~ H²0² + 0² Scheme 4

F e2 + + H202 - F e3 + + «OH + OH"

is the inactivating species exerting the deleterious effects independently o f its iron-catalyzed interaction w i t h H^O^

[64].

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