28
Biochimica et Biophysiea Acta, 542 (1978) 28--38 © Elsevier/North-Holland Biomedical Press
BBA 28587
SUPEROXIDE DISMUTASE OF THE EYE RELATIVE FUNCTIONS OF SUPEROXIDE DISMUTASE AND CATALASE IN PROTECTING THE OCULAR LENS FROM OXIDATIVE DAMAGE
KAILASH C. BHUYAN and D U R G A K. BHUYAN
Biochemistry Laboratory, The Eye-Bank for Sight Restoration, Inc., Manhattan Eye, Ear and Throat Hospital, 210 East 64 Street, New York, N.Y. 10021 (U.S.A.) (Received December 14th, 1977)
Summary 1. Activities of superoxide dismutase (superoxide : superoxide oxidoreductase, EC 1.15.1.1) have been estimated in eye tissues. In rabbit eye, superoxide dismutase is present in corneal epithelium, corneal endothelium, lens, iris, ciliary body and retina. In lens the major activity is in capsule epithelium. 2. Copper chelator diethyldithiocarbamate inhibited lens superoxide dismutase in vitro and in vivo in rabbit. 3. H202 caused inhibition of superoxide dismutase activity of lens extract, and this inhibition was potentiated by the catalase inhibitor 3-amino-Ill-I,2, 4-triazole (3-aminotriazole) or NAN3. 3-Aminotriazole or NaN3 had no effect on lens superoxide dismutase. Thus endogenous catalase of lens affords protection to the lens superoxide dismutase from inactivation by H202. 4. In rabbit having early cataract {vacuolar stage) induced by feeding-3aminotriazole, there was a decrease in superoxide dismutase of lens, a fall in ascorbic acid of ocular humors and lens, and a 2--3-fold increase in H202 of aqueous h u m o r and vitreous humor. We conclude that catalase of eye affords protection to the lens from H202 and it also protects superoxide dismutase of lens from inactivation by H202. Superoxide dismutase, in turn, protects the lens from the superoxide radical, O2'-. It is likely that inhibition of these enzymes may lead to production of the highly reactive oxidant, the h y d r o x y l radical, under pathological conditions when H202 concentration in vivo exceeds physiological limits as in cataract induced by 3-aminotriazole. A scheme of reaction mechanism has been proposed to explain the relative functions of ocular catalase and superoxide dismutase. Such a mechanism may be involved in cataractogenic process in the human.
29 Introduction Superoxide anion free radical (O2"-) is generated during some biological oxidations by univalent reduction of molecular oxygen [1]. It is considered at present that 02'-, its derivatives, H202, singlet oxygen (102) and OH- could be the primary source of oxidative damage in the cell [1]. Oxidative damage as the causative factor has been suggested in retrolental fibroplasia [ 2], and in cataract produced in mice by hyperbaric oxygen [3]. In cells exposed to X-ray or ultraviolet irradiation, oxygen-free radicals and H202 are produced [4,5] and these agents are cataractogenic [6,7]. A significant increase in H202 of aqueous humor and vitreous humor of rabbit was produced as a result of inhibition of catalase of eye tissues by 3-aminotriazole in vivo [8]. 3-Aminotriazole is a cataractogenic agent [9]. It has been proposed that free radicals or factors possessing the properties of free radicals may be the initiating agent in cataractogenesis [8,10,11]. Superoxide dismutase (superoxide : superoxide oxidoreductase, EC 1.15.1.1) protects the tissues from deleterious effects of 02"-, and by its activity produces H202 [12], O~.+O~.+2H*~
H202 + O 2 .
Evidence in the literature suggests that superoxide dismutase and the two H202metabolizing enzymes, catalase and peroxidase, may constitute an important defense mechanism against oxygen toxicity in cells [1,10,13]. In this communication we present some experimental results to elucidate the relative functions of ocular superoxide dismutase and catalase in protecting the crystalline lens from endogenous O2"- and H202. A brief report regarding this study has been made elsewhere [10]. Materials and Methods Chemicals. 1,2,3-Trihydroxybenzene (pyrogallol), diethylenetriamine pentaacetic acid, 2,6-dichlorophenolindophenol sodium salt (Grade 1), horseradish peroxidase (Type VI), L-ascorbic acid sodium salt, crystallized bovine serum albumin, crystalline 6 - h y d r o x y d o p a m i n e . HBr, horse heart c y t o c h r o m e c (Type III), xanthine oxidase (Grade 1), xanthine, diethyldithiocarbamic acid sodium salt and disodium ethylenediaminetetraacetate (Na2 • EDTA) were obtained from Sigma Chemical Co. Metaphosphoric acid, reagent grade, was from Mallinckrodt Chemical Works; 8-amino-l-naphthol-3,6-disulfonic acid monosodium salt of technical grade and 3-aminotriazole of practical grade were from Eastman Kodak Organic Chemicals; 3-aminotriazole, 98% pure (according to the manufacturer) was from Aldrich Chemical Co. and all other chemicals of certified or reagent grade were from Fisher Scientific Co. Test animals. Healthy Dutch rabbits of either sex weighing 1.5--2 kg with normal eyes were used for the in vitro study. For in vivo study, healthy, just weaned (4--6-week) littermate Dutch rabbits, weighing 0.4--0.7 kg with normal eyes were used to produce cataract by paired feeding of 3-aminotriazole in diet. The special diet was prepared by Farmers Cooperative Association of Vineland, Inc., Norma, N.J. by mixing 1.8 kg 3-aminotriazole with 454 kg of regular formula of rabbit chow prior to pelleting. 3-Aminotriazole content as estimated in
30 the repelleted special diet was 0.26%, showing that there was some loss during processing. In the littermate pair, the control rabbit was fed daily with regular rabbit chow equal to the a m o u n t of special food eaten by the experimental rabbit during the previous 24 h. Both groups received tap water for drinking. Slitlamp biomicroscopic examination of the control and experimental rabbit eyes under full mydriasis was done routinely to observe development of cataract. Early signs of cataract were observed after 2--4 weeks of feeding special diet. Such lenses were used as the model of early cataract for in vivo study. Preparation o f e n z y m e extracts. Rabbits were killed by air embolism, eyes were enucleated, washed with physiological saline (0.15 M NaC1)and dissected for cornea and lens. Prior to the dissection of vascularized eye tissues such as iris, cilliary body and retina, the rabbit was given general anaesthesia by intravenous sodium pentobarbital and perfused with 1 1 physiological saline via both carotid arteries for approx. 1 h to remove as much blood as possible. Fresh calf eyes, kept on ice, were received within 6--8 h of death of the animal from Max Insel Cohen of Livingston, N.J. Normal eyes were selected, washed with cold physiological saline, and lenses, lens capsule epithelium and decapsulated lenses were taken for the preparation of enzyme extracts. Normal human lenses were obtained from the donor eyes received at the Corneal Laboratory of The Eye-Bank for Sight Restoration, Inc., within 24 h of death. Lenses were from the age group 13--44 years. Using 50 mM phosphate buffer, pH 7.2, as an extractant, homogenates were prepared from corneal epithelium, corneal endothelium, lens, lens capsuleepithelium, decapsulated lens, iris, ciliary body and retina as described previously [8]; 750 × g supernatant of tissue homogenate was dialysed overnight against 5 m M phosphate buffer, pH 7.2, at 0--4°C and centrifuged at 39 000 Xg for 20 min. The supernatant obtained was used as the source of enzyme. E n z y m e assays. Superoxide dismutase activity was determined spectrophotometrically by the technique of Marklund and Marklund [14] which is based on the ability of this enzyme to inhibit 02"--dependent autoxidation of pyrogallol. The rate of autoxidation was measured by the increase in absorbance at 420 nm using Beckman DK-2A ratio-recording spectrophotometer. We compared this technique with that of McCord and Fridovich [12], which is based on the ability of this enzyme to inhibit the reduction of ferricytochrome c by 02" produced by the xanthine-xanthine oxidase system. Assay of purified bovine blood superoxide dismutase (Sigma) by both techniques gave 84--95% recovery, and the activities of lens and corneal epithelium as determined by two methods gave similar results. In order to study the effect of diethyldithiocarbamate on lens superoxide dismutase in vitro and in vivo, we used the method described by Heikkila et al. [15] to estimate the enzyme activity, since this c o m p o u n d even at low concentration was found to interfere with both the techniques mentioned above. Estimation o f ascorbic acid, H202 and 3-aminotriazole. Ascorbic acid and H202 in aqueous h u m o r and vitreous h u m o r were determined spectrophotometrically [8]. A protein-free 3% metaphosphoric acid extract of lens (1 g wet lens/10 ml acid) was adjusted to pH 3.5--3.6 by citrate buffer (0.44 M citric acid in 0.8 M NaOH) and a suitable aliquot was taken for spectrophotometric
31 estimation of ascorbic acid at 510 nm using 2,6-dichlorophenolindophenol as described by Roe [16]. 3-Aminotriazole in aqueous humor, vitreous h u m o r and lens was assayed by the technique of Agrawal and Margoliash [17] as described previously [8]. Protein estimation. Protein of tissue extracts was determined by the m e t h o d of Lowry et al. [18] using crystallized bovine serum albumin as standard. Results
Superoxide dismutase in eye. Table I shows the distribution of superoxide dismutase activities in rabbit eye tissues, calf lens and human lens. Among the eye tissues, activities of superoxide dismutase of iris and ciliary body were highest. Retina, corneal epithelium and corneal endothelium had about half the activity of ciliary body. In lens capsule epithelium of rabbit and calf, activities were about 7 and 14 times that of the decapsulated lens, respectively. Superoxide dismutase of eye tissues was destroyed when the enzyme extracts were boiled at 100°C for 15 min. Besides superoxide dismutase, we have also observed the activities (unit/mg protein + S.D.) of catalase {0.048 + 0.018, n = 8) and GSH peroxidase (0.033 ± 0.007, n = 8) in normal h u m a n lenses. Superoxide dismutase, 3-aminotriazole and H202 in early cataract. Cataract was induced in rabbits by feeding a diet containing 3-aminotriazole. Early cataract (vacuolar stage) was produced after 2--4 weeks, as shown in Fig. 1. Occasionally, cataract could be observed as early as at the end of the first week. The slit-lamp photograph of a typical cataract shows clusters of subcapsular
TABLE
I
SUPEROXIDE
DISMUTASE
ACTIVITY
OF EYE
The assay system consisted of 0.4 mM pyrogallol, 0.05 mM diethylenetriamine pentaacetate, 50 mM Tris(hydroxymethyl)aminomethane. HC1 b u f f e r ( p H 8 . 2 ) a n d a n a p p r o p r i a t e a m o u n t o f e n z y m e e x t r a c t (prepared as described in the text under Materials and Methods), in a final volume of 1.5 ml at room temperature (approx. 25°C). One unit of superoxide dismutase represents the amount of enzyme that inhibits 50% of the rate of autoxidation of pyrogallol under the defined assay conditions. Source
Eye tissue
Superoxide dismutase (units/mg protein)
Rabbit
Iris Ciliary body Retina Corneal epithelium Corneal endothelium Lens Lens capsule epithelium Decapsulated lens
16.632 12.623 4.875 5.503 5.091 0.205 1.012 0.139
Calf
Lens Lens capsule epithelium Decapsulated lens
0.201 z 0.031 (10) ** 1.752 ± 0.240 (35) 0.124 ± 0.013 (10)
Human
Lens
0 . 3 8 0 +~ 0 . 1 1 8
* Mean ± S.D.; number * * M e a n -+ S . D . ; n u m b e r
of animals studied in parentheses. of lenses studied in parentheses.
~+ 2 . 2 4 9 ± 2.791 ~ 0.951 ~ 0.258 ~+ 0 . 2 3 2 -+ 0 . 0 3 0 ± 0.089 ± 0.011
activity
(7) * (7) (7) (7) (6) (4) (5) (3)
(8)**
32
F i g . 1. S l i t - l a m p p h o t o g r a p h of a rabbit cataractous lens showing a typical early stage (vacuolar stage). Clusters of subcapsular vacuoles are seen occupying the equatorial and the anterior peripheral parts of the l e n s . M a g n i f i c a t i o n X 15.
vacuoles in the equatorial and peripheral zone of the lens. Other findings which are not shown in the picture were separation of sutures, lamellar separation and vacuoles along the posterior suture lines. The detailed morphological aspects of this cataract have been reported earlier [9]. Superoxide dismutase activity in early cataract (Table II) was decreased to
33 T A B L E II SUPEROXIDE DISMUTASE IN EARLY CATARACT AND 3-AMINOTRIAZOLE IN L E N S A N D OCULAR HUMORS OF LITTERMATE RABBITS AFTER PAIRED FEEDING ON SPECIAL DIET A s s a y s y s t e m f o r s u p e r o x i d e d i s m u t a s e a c t i v i t y w a s as d e s c r i b e d in t h e c a p t i o n t o T a b l e I. 3 - A m i n o t r i a z o l e c o n c e n t r a t i o n in a q u e o u s h u m o r a n d 27 0 0 0 X g s u p e r n a t a n t o b t a i n e d f r o m t r i c h l o r o a c e t i c a c i d e x t r a c t o f l e n s or v i t r e o u s h u m o r w a s d e t e r m i n e d b y d i a z o t i z a t i o n o f 3 - a m i n o g r o u p f o l l o w e d b y c o u p l i n g w i t h 8-amino-l-naphthol-3,6-disulfonic acid. Rabbit
Control on regular diet f o r 2--4 w e e k s E x p e r i m e n t a l on special diet for 2 weeks 2--4 weeks 4 weeks
Superoxide dismutase activity
3 - A m i n o t r i a z o l e c o n c e n t r a t i o n **
Lens (unit/mg protein)
Aqueous humor (mM)
Lens (pmol/g wet wt.)
Vitreous humor (mM)
0 . 2 4 5 -+ 0 . 0 0 3 (4) *
0.0 (18)
0.0 (9)
0.0 (15)
0 . 1 6 2 + 0 . 0 0 8 (6)
0 . 1 7 -+ 0 . 0 6 ( 1 2 ) -0 . 1 4 -+ 0 . 0 4 ( 6 )
0 . 2 2 + 0 . 0 1 (3) -0 . 1 2 -+ 0 . 0 4 (6)
0 . 1 5 -+ 0 . 0 8 ( 1 2 ) -0.12 2 0.04 (3)
* M e a n + S.D.; n u m b e r o f l e n s e s in p a r e n t h e s e s . ** M e a n -+ S.D.; n u m b e r o f r a b b i t s s t u d i e d in p a r e n t h e s e s .
TABLE III H202 OF AQUEOUS HUMOR AND PAIRED FEEDING ON SPECIAL DIET
VITREOUS
HUMOR
IN
LITTERMATE
RABBITS
AFTER
A q u e o u s h u m o r , w a s d i r e c t l y t a k e n f o r a s s a y . V i t r e o u s h u m o r w a s a s p i r a t e d b y s y r i n g e , c e n t r i f u g e d at 12 0 0 0 X g a n d s u p e r n a t a n t w a s u s e d f o r a s s a y . I n a silica e u v e t t e o f 1 - c m l i g h t p a t h , 3 m l o f k n o w n c o n c e n t r a t i o n ( a b o u t 0 . 0 4 m M ) o f 2 , 6 - d i c h l o r o p h e n o l i n d o p h e n o l in 50 m M p h o s p h a t e b u f f e r ( p H 6 . 6 ) w a s t a k e n ; 5 0 - - 1 0 0 pl o f a q u e o u s h u m o r o r 1 0 0 - - 2 0 0 /~l o f t h e 12 0 0 0 X g s u p e r n a t a n t o f v i t r e o u s h u m o r w a s a d d e d , m i x e d a n d a s t e a d y fall in a b s o r b a n c e at 6 1 0 n m d u e t o r e d u c t i o n o f t h e d y e b y a s c o r b i c a c i d p r e s e n t in t h e s a m p l e w a s m e a s u r e d . A f e w pl o f h o r s e r a d i s h p e r o x i d a s e ( T y p e V I S i g m a , 5 m g ] m l p h o s p h a t e b u f f e r ) w e r e t h e n a d d e d a n d a n i n c r e a s e i n a b s o r b a n c e at 6 1 0 n m d u e to r e o x i d a t i o n o f t h e leuco d y e b y H 2 0 2 p r e s e n t in t h e s a m p l e w a s m e a s u r e d . F r o m t h e s t o i c h i o m e t r y o f t h e r e a c t i o n s i n v o l v e d , H 2 0 2 in a q u e o u s h u m o r a n d v i t r e o u s h u m o r w a s c a l c u l a t e d u s i n g e 6 1 0 n m o x i d i z e d d y e = 21 0 0 0 . Rabbit
H202 concentration Aqueous humor (mM)
Control on regular diet E x p e r i m e n t a l on special diet for 2 weeks Control on regular diet E x p e r i m e n t a l on special diet for 4 weeks
Vitreous humor Percent control
0 . 0 5 7 -+ 0 . 0 1 5 (4) *
0 . 1 1 6 -+ 0 . 0 0 6 ( 4 )
Percent control
0 . 0 2 5 -+ 0 . 0 0 7 ( 4 )
204
0 . 0 6 0 -+ 0 . 0 0 8 ( 5 )
0 . 1 7 0 -+ 0 . 1 4 1 ( 5 )
(raM)
0 . 0 5 5 -+ 0 . 0 0 3 (4)
220
0.018 + 0.007 (5)
283
* M e a n -+ S.D.; n u m b e r o f r a b b i t s s t u d i e d in p a r e n t h e s e s .
0 . 0 3 9 -+ 0 . 0 3 1 ( 5 )
217
34 TABLE
IV
ASCORBIC ACID OF AQUEOUS BITS AFTER PAIRED FEEDING
HUMOR, LENS AND ON SPECIAL DIET
VITREOUS
HUMOR
IN LITTERMATE
RAB-
Aqueous humor (50--100 pl) or 12 000 × g supernatant of vitreous humor (100--200 pl) was added to 3 ml of 2,6-dichlorophenolindophenol of known concentration ( a b o u t 0 . 0 4 r a M ) in 5 0 m M p h o s p h a t e b u f fer (pH 6.6) at room temperature (approx. 25°C): mixed, and the decrease in absorbance due to reduction of the dye was measured at 610 nm. From the stoichiometry of the reactions involved, ascorbic acid cone e n t r a t i o n o f a q u e o u s h u m o r a n d v i t r e o u s h u m o r w a s d e t e r m i n e d f r o m c 6 1 0 n m o x i d i z e d d y e = 21 0 0 0 . T e c h n i q u e u s e d f o r e s t i m a t i o n o f a s c o r b i c a c i d o f l e n s is d e s c r i b e d u n d e r M a t e r i a l s a n d M e t h o d s in t h e text. Rabbit
Ascorbic acid concentration Aqueous (mM)
humor
Lens (pmol/g
wet wt.)
Vitreous humor (mM)
Control on regular diet
1.072 + 0.322 (6) *
1.183 ± 0.071
(4)
0.416 ± 0.096 (6)
Experimental on special diet for 2 weeks
0.380 ± 0.080 (6)
0.872 ± 0.071
(6)
0.145 ± 0.071 (6)
Control on regular diet
1.200 ± 0.390 (6)
1.120 ± 0.169 (10)
0.308 ± 0.091 (6)
Experimental on special diet for 4 weeks
0.502 ± 0.214 (6)
0.652 ± 0.078
0.111 ± 0.049 (6)
* Mean ± S.D.; number
(8)
of rabbits studied in parentheses.
0.20 o
o.
,~
O.15
~
O.lO
I1
~
o.05
m
o.0
J
I ~
0.05
O.10
h 0.50
m M H202 F i g . 2. E f f e c t o f H 2 0 2 o n s u p e r o x i d e d i s m u t a s e o f c a l f l e n s in v i t r o . T h e a s s a y s y s t e m h a s b e e n d e s c r i b e d i n c a p t i o n t o T a b l e I. H 2 0 2 ( o e), H202 and 20 mM 3-aminotriazole (X------×) and H202 and 1 mM NaN 3 (o-------o) were added to the assay system, incubated at room temperature (about 25°C) for 10 rain and superoxide dismutase reaction was initiated by pyrogallol. When 3-aminotriazole was used, l e n s e x t r a c t w a s p r e i n c u b a t e d w i t h it f o r 1 h a n d t h e n t a k e n f o r a s s a y . A n a v e r a g e o f 3 - - 4 e s t i m a t i o n s is presented.
35
0.20 .>
o
,_~ 5:
0.15
~"
0.10
\
0.00
t
I
|
I
O.0
0.25
0.50
0.75
I
,,.
1.OO
g Diethyldithiocarbamate injected/kg body wt Fig. 3. E f f e c t o f d i e t h y l d i t h i o c a r b a m a t e on s u p e r o x i d e d i s m u t a s e of r a b b i t lens in vivo. D i e t h y l d i t h i o c a r b a m a t e d i s s o l v e d i n p h y s i o l o g i c a l s a l i n e w a s a d m i n i s t e r e d i n t r a v e n o u s l y t o t h e e x p e r i m e n t a l r a b b i t as a single dose. The c o n t r o l r a b b i t received an i n t r a v e n o u s i n j e c t i o n of e q u a l v o l u m e of p h y s i o l o g i c a l saline. Assay system consisted of 0.2 mM 6-hydroxydopamine, 50 m M K 2 H P O 4 / K H 2 P O 4 b u f f e r (pH 6.5), 0.1 m M N a 2 • E D T A a n d an a p p r o p r i a t e a m o u n t o f e n z y m e e x t r a c t ( p r e p a r e d f r o m l e n s as d e s c r i b e d u n d e r Materials a n d M e t h o d s in the t e x t ) in a final v o l u m e of 1.5 m l at r o o m t e m p e r a t u r e ( a p p r o x . 2 5 ° C ) . O n e unit of superoxide dismutase produced 50% inhibition of the rate of autoxidation of 6-hydroxydopamine as m e a s u r e d b y i n c r e a s e i n a b s o r b a n c e a t 4 9 0 n m . A c t i v i t y ( U n i t s / m g p r o t e i n ) m e a n + S . D . , n = 5 c o n t r o l r a b b i t s a n d a v e r a g e a c t i v i t y o f t h e l e n s e s o f e a c h e x p e r i m e n t a l r a b b i t is p l o t t e d .
66% of control. As estimated, after 2 weeks of feeding the special diet to rabbit, 3-aminotriazole was present in aqueous humor, lens and vitreous h u m o r (Table II). After 4 weeks, the concentration of 3-aminotriazole in eye humors decreased by 20% and in lens by 47%. Table III shows that in rabbit having early cataract, H202 of aqueous h u m o r and vitreous h u m o r was increased by approx. 2--3 times the control. Ascorbic acid in eye humors and lens o f rabbit having early cataract. The data in Table IV show that ascorbic acid of aqueous h u m o r was decreased to approx. 40% of control, vitreous h u m o r to approx. 36% of control, and lens to 58--74% of control, suggesting a fall in the antioxygenic potential of lens. Effect o f H202 on the superoxide dismutase o f calf lens in vitro. Fig. 2 shows that H202 inhibits superoxide dismutase of lens extract. H202 (0.05 mM) caused approx. 8% inhibition which increased to 22--32% in the presence of 20 mM 3-aminotriazole or 1 mM NAN3. A 25% inhibition of superoxide dismutase produced by 0.5 mM H202 was increased to approx. 56% when 3-aminotriazole or NaN3 was added. 20 mM 3-aminotriazole or 1 mM NaN3 produced no effect on lenticular superoxide dismutase. 3-Aminotriazole is a specific inhibitor of catalase [19] and NaN3 is also a p o t e n t inhibitor of this enzyme. Thus, when the protective effect of endogenous catalase of lens was abolished, the inhibitory effect of H2Oz on lens superoxide dismutase was enhanced. Effect o f diethyldithiocarbamate on superoxide dismutase o f lens in vitro and in vivo. Incubation of rabbit lens extract with 1 mM diethyldithiocarbamate produced 40% inhibition of superoxide dismutase activity. Fig. 3 shows that a single dose of 1 g diethyldithiocarbamate/kg b o d y weight in rabbit caused 84% inhibition of lens superoxide dismutase 2.5 h after injection. By reducing the dose to 0.7 g/kg b o d y weight, approx. 50% inhibition was produced after 2.75 h. Further reduction of the dose to 0.5 g/kg b o d y weight
36 resulted in 27% inhibition at 3 h after injection. Our experience with this compound in vivo is not very encouraging because with a dose which causes approx. 50--80% inhibition of superoxide dismutase, the rabbit hardly survives up to 3 h. By reducing the dose to 0.5 g/kg b o d y weight, diethyldithiocarbamate does not cause significant inhibition of enzyme at 3 h after injection. Discussion
Experimental evidence provided here demonstrates the participation of superoxide dismutase and catalase in protecting the crystalline lens from oxidative damage by O2"- and H202. In eye, superoxide dismutase has been previously shown [20] in retinal rod outer segment. We observed that this enzyme is also present in iris, ciliary body, retina, corneal epithelium, corneal endothelium and lens of rabbit and in the lenses of calf and the human. NaCN (1 mM) produced 50% inhibition of superoxide dismutase activity of calf lens extract suggesting that it could be a Cu 2÷- and/or Zn2+-containing enzyme. Further indication a b o u t the Cu 2÷ nature of the lenticular superoxide dismutase was shown b y its inhibition by diethyldithiocarbamate in vitro and in vivo. This c o m p o u n d has been shown [15] to inhibit superoxide dismutase of blood, liver and brain of mice in vivo. H202 has been shown to inactivate (Cu 2÷ + Zn 2÷)-superoxide dismutase [21]. Superoxide dismutase activity of calf lens extract was inhibited by H202 and catalase inhibitor, 3-aminotriazole or NaN3 was found to potentiate this inhibitory effect of H202 by 2--4-fold. In early cataract induced in rabbits b y feeding a diet containing 3-aminotriazole, H20~ concentrations in aqueous h u m o r and vitreous h u m o r were increased by 2--3-fold and superoxide dismutase activity of cataractous lens was decreased. Thus, it is conceivable that in cataract, increased H202 of ocular humors causes inactivation of superoxide dismutase of lens in vivo. The fall in ascorbic acid of aqueous humor, vitreous humor and lens which we observed in early cataract may be due to its oxidation b y O2"-. Oxidation of ascorbic acid by O5"- has been reported [22]. As illustrated in the following scheme (Fig. 4), we propose the enzymatic and nonenzymatic reaction mechanism which may be involved in cataractogenesis. Hydrogen peroxide and molecular oxygen are produced during the disproportionation of the O2'- by superoxide dismutase [12]. Catalase and peroxidase protect the tissues from the toxic effects of H202 [23] and both enzymes are present in eye tissues [8,24]. Selective inhibition of catalase of iris, ciliary b o d y , retina, cornea and lens by 3-aminotriazole resulted in significant increase in H202 of ocular humors of rabbit in vivo. However, under the conditions unaltered GSH peroxidase of eye tissues alone failed to regulate H~O2 in ocular humors within the physiological limit [8]. The increased H202 inactivates superoxide dismutase of lens and possibly of other eye tissues. Thus the enzymatic defense against the toxicity of O5"- being impaired, H202 and 05"could further react, possibly by the Haber-Weiss reaction [25], producing OHwhen H202 concentration exceeds the physiological limit under pathological conditions as in cataract induced by 3-aminotriazole. Besides H202 and O2'- it seems likely that OH-, the most p o t e n t oxidant known, could be the ultimate initiator of cataractogenic process.
37 x
02+
,x Oe"
O~
02: +
2H +
~S,~oo g/ X
H202.1. H202 -t-
Catalase ~2H20 .i. 02
x
Fig. 4. S c h e m a t i c r e p r e s e n t a t i o n of t h e e n z y m a t i c a n d n o n e n z y m a t i c r e a c t i o n s w h i c h m a y b e i n v o l v e d in the m e c h a n i s m of c a t a r a c t o g e n e s i s . SOD, s u p e r o x i d e d i s m u t a s e , AH2, h y d r o g e n d o n o r a n d A, o x i d i z e d d o n o r . D e t a i l e d e x p l a n a t i o n f o l l o w s in t h e t e x t .
To conclude, catalase of eye affords protection to the lens from H202 and it also protects superoxide dismutase of lens from the inhibitory effect of H202. Superoxide dismutase, in turn, protects the lens from 02"- and it is possible that b o t h superoxide dismutase and catalase might prevent the formation of
OH.. Acknowledgements This investigation was supported by NIH research grant No. EY01731 from the National Eye Institute, Department of Health, Education and Welfare, United States Public Health Service. Our sincere thanks are due to Dr. Harold L. Kern for going through the manuscript and for his helpful suggestions and Dr. Arnold I. Turtz, Dr. R. David Sudarsky and Dr. Herbert M. Katzin for their generous encouragement. We dedicate this paper to Professor L.P. Agarwal and Professor R.K. Mishra.
References 1 F r i d o v i c h , 1. ( 1 9 7 6 ) in F r e e R a d i c a l s in Biology ( P r y o r , W.A., ed.), Vol. 1, pp. 2 3 9 - - 2 7 1 , A c a d e m i c Press, N e w Y o r k 2 K i n s e y , V.E., J a c o b u s , J.T. a n d H e m p h i l l , F.M. ( 1 9 5 6 ) A r c h . O p h t h a l m o l . 5 6 , 4 8 1 - - 5 4 3 3 S e h o c k e t , S.S., E s t e r s o n , J., B r a d f o r d , B., Michaelis, M. a n d R i c h a r d s , R.D. ( 1 9 7 2 ) Israel J. Med. Sci. 8, 1 5 9 6 - - 1 5 9 9 4 Weiss, J. ( 1 9 4 6 ) N a t u r e 157, 584 5 L a t a r j e t , R. and Caldas, L.R. ( 1 9 5 5 ) J. Gen. Physiol. 35, 4 5 5 - - 4 7 0 6 V o n S a l l m a n , L. ( 1 9 5 1 ) A r c h . O p h t h a l m o l . ( C h i c a g o ) 45, 1 4 9 - - 1 6 4 7 Z i g m a n , S., S c h u l t z , J. a n d Y u l o , T. ( 1 9 7 3 ) E x p . E y e Res. 15, 2 0 1 - - 2 0 8 8 B h u y a n , K.C. and B h u y a n , D.K. ( 1 9 7 7 ) B i o c h i m . B i o p h y s . A c t a 497, 6 4 1 - - 6 5 1 9 B h u y a n , K . C . , B h u y a n , D . K . a n d K a t z i n , H.M. ( 1 9 7 3 ) O p h t h a l m i c Res. 5, 2 3 6 - - 2 4 7 10 B h u y a n , K.C. a n d B h u y a n , D.K. ( 1 9 7 7 ) I.R.C.S. Med. Sci. 5, 279
38 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
L e r m a n , S., K u c k , Jr., J.F., B o r k m a n , R.F. a n d Saker, E. ( 1 9 7 6 ) O p h t h a l m i c Res. 8, 213 22(; McCord, J.M. and F r i d o v i c h , I. ( 1 9 6 9 ) J. Biol. C h e m . 244, 6 0 4 9 - 6 0 5 5 Hassan, H.M. and F r i d o v i c h , I. ( 1 9 7 7 ) J. Bacteriol. 129, 1 5 7 4 1 5 8 3 M a r k l u n d , S. and M a r k l u n d , G. ( 1 9 7 4 ) Eur. J. B i o c h e m . 47, 4 6 9 - - 4 7 4 Heikkila, R.E., C a b b a t , F.S. a n d C o h e n , G. ( 1 9 7 6 ) J . Biol. C h e m , 251, 2 1 8 2 . - 2 1 8 5 Roe, J.H. ( 1 9 5 4 ) in M e t h o d s of B i o c h e m i c a l Analysis (Glick, D., ed.), Vol, 1, 1 1 6 - - 1 3 9 , I n t e r s e i e n c e Publ, A g r a w a l , B.B.L. and Margoliash, E. ( 1 9 7 0 ) AnM. B i o e h e m . 34, 5 0 5 - - 5 1 6 L o w r y , O.H,, R o s e b r o u g h , N.J., Farr, A.L. and Randall, R.J. ( 1 9 5 1 ) J. Biol. C h e m . 193, 265 2 7 5 t t e i m , W.G., A p p l e m a n , D. a n d P y f r o m , H.T. ( 1 9 5 6 ) A m . J. Physiol. 186, 1 9 - - 2 3 H a l l M.O. and Hall, D.O. ( 1 9 7 5 ) B i o e h e m . Biophys. IRes. C o m m u n . 67, 1199 1 2 0 4 Rotilio, G., M o r p u r g o , L., Calabrese, L. a n d M o n d o v i , B. ( 1 9 7 3 ) B i o e h i m . Biophys. A c t a 302, 2 2 9 - 235 Nishikimi, M. ( 1 9 7 5 ) B i o c h e m . Biophys. Res. C o m m u n . 63, ,163--468 Nieholls, P. ( 1 9 7 2 ) B i o c h i m . Biophys. A e t a 279, 306---309 Pirie, A. ( 1 9 6 5 ) B i o e h e m . J. 96, 2 4 4 253 H a b e r , F. a n d Weiss, d. ( 1 9 3 4 ) Proe. R. Soc. L o n d , , Ser. A 147, 3 3 2 - - 3 5 1
Biochimica et Biophysiea Acta, 542 (1978) 28--38 © Elsevier/North-Holland Biomedical Press
BBA 28587
SUPEROXIDE DISMUTASE OF THE EYE RELATIVE FUNCTIONS OF SUPEROXIDE DISMUTASE AND CATALASE IN PROTECTING THE OCULAR LENS FROM OXIDATIVE DAMAGE
KAILASH C. BHUYAN and D U R G A K. BHUYAN
Biochemistry Laboratory, The Eye-Bank for Sight Restoration, Inc., Manhattan Eye, Ear and Throat Hospital, 210 East 64 Street, New York, N.Y. 10021 (U.S.A.) (Received December 14th, 1977)
Summary 1. Activities of superoxide dismutase (superoxide : superoxide oxidoreductase, EC 1.15.1.1) have been estimated in eye tissues. In rabbit eye, superoxide dismutase is present in corneal epithelium, corneal endothelium, lens, iris, ciliary body and retina. In lens the major activity is in capsule epithelium. 2. Copper chelator diethyldithiocarbamate inhibited lens superoxide dismutase in vitro and in vivo in rabbit. 3. H202 caused inhibition of superoxide dismutase activity of lens extract, and this inhibition was potentiated by the catalase inhibitor 3-amino-Ill-I,2, 4-triazole (3-aminotriazole) or NAN3. 3-Aminotriazole or NaN3 had no effect on lens superoxide dismutase. Thus endogenous catalase of lens affords protection to the lens superoxide dismutase from inactivation by H202. 4. In rabbit having early cataract {vacuolar stage) induced by feeding-3aminotriazole, there was a decrease in superoxide dismutase of lens, a fall in ascorbic acid of ocular humors and lens, and a 2--3-fold increase in H202 of aqueous h u m o r and vitreous humor. We conclude that catalase of eye affords protection to the lens from H202 and it also protects superoxide dismutase of lens from inactivation by H202. Superoxide dismutase, in turn, protects the lens from the superoxide radical, O2'-. It is likely that inhibition of these enzymes may lead to production of the highly reactive oxidant, the h y d r o x y l radical, under pathological conditions when H202 concentration in vivo exceeds physiological limits as in cataract induced by 3-aminotriazole. A scheme of reaction mechanism has been proposed to explain the relative functions of ocular catalase and superoxide dismutase. Such a mechanism may be involved in cataractogenic process in the human.
29 Introduction Superoxide anion free radical (O2"-) is generated during some biological oxidations by univalent reduction of molecular oxygen [1]. It is considered at present that 02'-, its derivatives, H202, singlet oxygen (102) and OH- could be the primary source of oxidative damage in the cell [1]. Oxidative damage as the causative factor has been suggested in retrolental fibroplasia [ 2], and in cataract produced in mice by hyperbaric oxygen [3]. In cells exposed to X-ray or ultraviolet irradiation, oxygen-free radicals and H202 are produced [4,5] and these agents are cataractogenic [6,7]. A significant increase in H202 of aqueous humor and vitreous humor of rabbit was produced as a result of inhibition of catalase of eye tissues by 3-aminotriazole in vivo [8]. 3-Aminotriazole is a cataractogenic agent [9]. It has been proposed that free radicals or factors possessing the properties of free radicals may be the initiating agent in cataractogenesis [8,10,11]. Superoxide dismutase (superoxide : superoxide oxidoreductase, EC 1.15.1.1) protects the tissues from deleterious effects of 02"-, and by its activity produces H202 [12], O~.+O~.+2H*~
H202 + O 2 .
Evidence in the literature suggests that superoxide dismutase and the two H202metabolizing enzymes, catalase and peroxidase, may constitute an important defense mechanism against oxygen toxicity in cells [1,10,13]. In this communication we present some experimental results to elucidate the relative functions of ocular superoxide dismutase and catalase in protecting the crystalline lens from endogenous O2"- and H202. A brief report regarding this study has been made elsewhere [10]. Materials and Methods Chemicals. 1,2,3-Trihydroxybenzene (pyrogallol), diethylenetriamine pentaacetic acid, 2,6-dichlorophenolindophenol sodium salt (Grade 1), horseradish peroxidase (Type VI), L-ascorbic acid sodium salt, crystallized bovine serum albumin, crystalline 6 - h y d r o x y d o p a m i n e . HBr, horse heart c y t o c h r o m e c (Type III), xanthine oxidase (Grade 1), xanthine, diethyldithiocarbamic acid sodium salt and disodium ethylenediaminetetraacetate (Na2 • EDTA) were obtained from Sigma Chemical Co. Metaphosphoric acid, reagent grade, was from Mallinckrodt Chemical Works; 8-amino-l-naphthol-3,6-disulfonic acid monosodium salt of technical grade and 3-aminotriazole of practical grade were from Eastman Kodak Organic Chemicals; 3-aminotriazole, 98% pure (according to the manufacturer) was from Aldrich Chemical Co. and all other chemicals of certified or reagent grade were from Fisher Scientific Co. Test animals. Healthy Dutch rabbits of either sex weighing 1.5--2 kg with normal eyes were used for the in vitro study. For in vivo study, healthy, just weaned (4--6-week) littermate Dutch rabbits, weighing 0.4--0.7 kg with normal eyes were used to produce cataract by paired feeding of 3-aminotriazole in diet. The special diet was prepared by Farmers Cooperative Association of Vineland, Inc., Norma, N.J. by mixing 1.8 kg 3-aminotriazole with 454 kg of regular formula of rabbit chow prior to pelleting. 3-Aminotriazole content as estimated in
30 the repelleted special diet was 0.26%, showing that there was some loss during processing. In the littermate pair, the control rabbit was fed daily with regular rabbit chow equal to the a m o u n t of special food eaten by the experimental rabbit during the previous 24 h. Both groups received tap water for drinking. Slitlamp biomicroscopic examination of the control and experimental rabbit eyes under full mydriasis was done routinely to observe development of cataract. Early signs of cataract were observed after 2--4 weeks of feeding special diet. Such lenses were used as the model of early cataract for in vivo study. Preparation o f e n z y m e extracts. Rabbits were killed by air embolism, eyes were enucleated, washed with physiological saline (0.15 M NaC1)and dissected for cornea and lens. Prior to the dissection of vascularized eye tissues such as iris, cilliary body and retina, the rabbit was given general anaesthesia by intravenous sodium pentobarbital and perfused with 1 1 physiological saline via both carotid arteries for approx. 1 h to remove as much blood as possible. Fresh calf eyes, kept on ice, were received within 6--8 h of death of the animal from Max Insel Cohen of Livingston, N.J. Normal eyes were selected, washed with cold physiological saline, and lenses, lens capsule epithelium and decapsulated lenses were taken for the preparation of enzyme extracts. Normal human lenses were obtained from the donor eyes received at the Corneal Laboratory of The Eye-Bank for Sight Restoration, Inc., within 24 h of death. Lenses were from the age group 13--44 years. Using 50 mM phosphate buffer, pH 7.2, as an extractant, homogenates were prepared from corneal epithelium, corneal endothelium, lens, lens capsuleepithelium, decapsulated lens, iris, ciliary body and retina as described previously [8]; 750 × g supernatant of tissue homogenate was dialysed overnight against 5 m M phosphate buffer, pH 7.2, at 0--4°C and centrifuged at 39 000 Xg for 20 min. The supernatant obtained was used as the source of enzyme. E n z y m e assays. Superoxide dismutase activity was determined spectrophotometrically by the technique of Marklund and Marklund [14] which is based on the ability of this enzyme to inhibit 02"--dependent autoxidation of pyrogallol. The rate of autoxidation was measured by the increase in absorbance at 420 nm using Beckman DK-2A ratio-recording spectrophotometer. We compared this technique with that of McCord and Fridovich [12], which is based on the ability of this enzyme to inhibit the reduction of ferricytochrome c by 02" produced by the xanthine-xanthine oxidase system. Assay of purified bovine blood superoxide dismutase (Sigma) by both techniques gave 84--95% recovery, and the activities of lens and corneal epithelium as determined by two methods gave similar results. In order to study the effect of diethyldithiocarbamate on lens superoxide dismutase in vitro and in vivo, we used the method described by Heikkila et al. [15] to estimate the enzyme activity, since this c o m p o u n d even at low concentration was found to interfere with both the techniques mentioned above. Estimation o f ascorbic acid, H202 and 3-aminotriazole. Ascorbic acid and H202 in aqueous h u m o r and vitreous h u m o r were determined spectrophotometrically [8]. A protein-free 3% metaphosphoric acid extract of lens (1 g wet lens/10 ml acid) was adjusted to pH 3.5--3.6 by citrate buffer (0.44 M citric acid in 0.8 M NaOH) and a suitable aliquot was taken for spectrophotometric
31 estimation of ascorbic acid at 510 nm using 2,6-dichlorophenolindophenol as described by Roe [16]. 3-Aminotriazole in aqueous humor, vitreous h u m o r and lens was assayed by the technique of Agrawal and Margoliash [17] as described previously [8]. Protein estimation. Protein of tissue extracts was determined by the m e t h o d of Lowry et al. [18] using crystallized bovine serum albumin as standard. Results
Superoxide dismutase in eye. Table I shows the distribution of superoxide dismutase activities in rabbit eye tissues, calf lens and human lens. Among the eye tissues, activities of superoxide dismutase of iris and ciliary body were highest. Retina, corneal epithelium and corneal endothelium had about half the activity of ciliary body. In lens capsule epithelium of rabbit and calf, activities were about 7 and 14 times that of the decapsulated lens, respectively. Superoxide dismutase of eye tissues was destroyed when the enzyme extracts were boiled at 100°C for 15 min. Besides superoxide dismutase, we have also observed the activities (unit/mg protein + S.D.) of catalase {0.048 + 0.018, n = 8) and GSH peroxidase (0.033 ± 0.007, n = 8) in normal h u m a n lenses. Superoxide dismutase, 3-aminotriazole and H202 in early cataract. Cataract was induced in rabbits by feeding a diet containing 3-aminotriazole. Early cataract (vacuolar stage) was produced after 2--4 weeks, as shown in Fig. 1. Occasionally, cataract could be observed as early as at the end of the first week. The slit-lamp photograph of a typical cataract shows clusters of subcapsular
TABLE
I
SUPEROXIDE
DISMUTASE
ACTIVITY
OF EYE
The assay system consisted of 0.4 mM pyrogallol, 0.05 mM diethylenetriamine pentaacetate, 50 mM Tris(hydroxymethyl)aminomethane. HC1 b u f f e r ( p H 8 . 2 ) a n d a n a p p r o p r i a t e a m o u n t o f e n z y m e e x t r a c t (prepared as described in the text under Materials and Methods), in a final volume of 1.5 ml at room temperature (approx. 25°C). One unit of superoxide dismutase represents the amount of enzyme that inhibits 50% of the rate of autoxidation of pyrogallol under the defined assay conditions. Source
Eye tissue
Superoxide dismutase (units/mg protein)
Rabbit
Iris Ciliary body Retina Corneal epithelium Corneal endothelium Lens Lens capsule epithelium Decapsulated lens
16.632 12.623 4.875 5.503 5.091 0.205 1.012 0.139
Calf
Lens Lens capsule epithelium Decapsulated lens
0.201 z 0.031 (10) ** 1.752 ± 0.240 (35) 0.124 ± 0.013 (10)
Human
Lens
0 . 3 8 0 +~ 0 . 1 1 8
* Mean ± S.D.; number * * M e a n -+ S . D . ; n u m b e r
of animals studied in parentheses. of lenses studied in parentheses.
~+ 2 . 2 4 9 ± 2.791 ~ 0.951 ~ 0.258 ~+ 0 . 2 3 2 -+ 0 . 0 3 0 ± 0.089 ± 0.011
activity
(7) * (7) (7) (7) (6) (4) (5) (3)
(8)**
32
F i g . 1. S l i t - l a m p p h o t o g r a p h of a rabbit cataractous lens showing a typical early stage (vacuolar stage). Clusters of subcapsular vacuoles are seen occupying the equatorial and the anterior peripheral parts of the l e n s . M a g n i f i c a t i o n X 15.
vacuoles in the equatorial and peripheral zone of the lens. Other findings which are not shown in the picture were separation of sutures, lamellar separation and vacuoles along the posterior suture lines. The detailed morphological aspects of this cataract have been reported earlier [9]. Superoxide dismutase activity in early cataract (Table II) was decreased to
33 T A B L E II SUPEROXIDE DISMUTASE IN EARLY CATARACT AND 3-AMINOTRIAZOLE IN L E N S A N D OCULAR HUMORS OF LITTERMATE RABBITS AFTER PAIRED FEEDING ON SPECIAL DIET A s s a y s y s t e m f o r s u p e r o x i d e d i s m u t a s e a c t i v i t y w a s as d e s c r i b e d in t h e c a p t i o n t o T a b l e I. 3 - A m i n o t r i a z o l e c o n c e n t r a t i o n in a q u e o u s h u m o r a n d 27 0 0 0 X g s u p e r n a t a n t o b t a i n e d f r o m t r i c h l o r o a c e t i c a c i d e x t r a c t o f l e n s or v i t r e o u s h u m o r w a s d e t e r m i n e d b y d i a z o t i z a t i o n o f 3 - a m i n o g r o u p f o l l o w e d b y c o u p l i n g w i t h 8-amino-l-naphthol-3,6-disulfonic acid. Rabbit
Control on regular diet f o r 2--4 w e e k s E x p e r i m e n t a l on special diet for 2 weeks 2--4 weeks 4 weeks
Superoxide dismutase activity
3 - A m i n o t r i a z o l e c o n c e n t r a t i o n **
Lens (unit/mg protein)
Aqueous humor (mM)
Lens (pmol/g wet wt.)
Vitreous humor (mM)
0 . 2 4 5 -+ 0 . 0 0 3 (4) *
0.0 (18)
0.0 (9)
0.0 (15)
0 . 1 6 2 + 0 . 0 0 8 (6)
0 . 1 7 -+ 0 . 0 6 ( 1 2 ) -0 . 1 4 -+ 0 . 0 4 ( 6 )
0 . 2 2 + 0 . 0 1 (3) -0 . 1 2 -+ 0 . 0 4 (6)
0 . 1 5 -+ 0 . 0 8 ( 1 2 ) -0.12 2 0.04 (3)
* M e a n + S.D.; n u m b e r o f l e n s e s in p a r e n t h e s e s . ** M e a n -+ S.D.; n u m b e r o f r a b b i t s s t u d i e d in p a r e n t h e s e s .
TABLE III H202 OF AQUEOUS HUMOR AND PAIRED FEEDING ON SPECIAL DIET
VITREOUS
HUMOR
IN
LITTERMATE
RABBITS
AFTER
A q u e o u s h u m o r , w a s d i r e c t l y t a k e n f o r a s s a y . V i t r e o u s h u m o r w a s a s p i r a t e d b y s y r i n g e , c e n t r i f u g e d at 12 0 0 0 X g a n d s u p e r n a t a n t w a s u s e d f o r a s s a y . I n a silica e u v e t t e o f 1 - c m l i g h t p a t h , 3 m l o f k n o w n c o n c e n t r a t i o n ( a b o u t 0 . 0 4 m M ) o f 2 , 6 - d i c h l o r o p h e n o l i n d o p h e n o l in 50 m M p h o s p h a t e b u f f e r ( p H 6 . 6 ) w a s t a k e n ; 5 0 - - 1 0 0 pl o f a q u e o u s h u m o r o r 1 0 0 - - 2 0 0 /~l o f t h e 12 0 0 0 X g s u p e r n a t a n t o f v i t r e o u s h u m o r w a s a d d e d , m i x e d a n d a s t e a d y fall in a b s o r b a n c e at 6 1 0 n m d u e t o r e d u c t i o n o f t h e d y e b y a s c o r b i c a c i d p r e s e n t in t h e s a m p l e w a s m e a s u r e d . A f e w pl o f h o r s e r a d i s h p e r o x i d a s e ( T y p e V I S i g m a , 5 m g ] m l p h o s p h a t e b u f f e r ) w e r e t h e n a d d e d a n d a n i n c r e a s e i n a b s o r b a n c e at 6 1 0 n m d u e to r e o x i d a t i o n o f t h e leuco d y e b y H 2 0 2 p r e s e n t in t h e s a m p l e w a s m e a s u r e d . F r o m t h e s t o i c h i o m e t r y o f t h e r e a c t i o n s i n v o l v e d , H 2 0 2 in a q u e o u s h u m o r a n d v i t r e o u s h u m o r w a s c a l c u l a t e d u s i n g e 6 1 0 n m o x i d i z e d d y e = 21 0 0 0 . Rabbit
H202 concentration Aqueous humor (mM)
Control on regular diet E x p e r i m e n t a l on special diet for 2 weeks Control on regular diet E x p e r i m e n t a l on special diet for 4 weeks
Vitreous humor Percent control
0 . 0 5 7 -+ 0 . 0 1 5 (4) *
0 . 1 1 6 -+ 0 . 0 0 6 ( 4 )
Percent control
0 . 0 2 5 -+ 0 . 0 0 7 ( 4 )
204
0 . 0 6 0 -+ 0 . 0 0 8 ( 5 )
0 . 1 7 0 -+ 0 . 1 4 1 ( 5 )
(raM)
0 . 0 5 5 -+ 0 . 0 0 3 (4)
220
0.018 + 0.007 (5)
283
* M e a n -+ S.D.; n u m b e r o f r a b b i t s s t u d i e d in p a r e n t h e s e s .
0 . 0 3 9 -+ 0 . 0 3 1 ( 5 )
217
34 TABLE
IV
ASCORBIC ACID OF AQUEOUS BITS AFTER PAIRED FEEDING
HUMOR, LENS AND ON SPECIAL DIET
VITREOUS
HUMOR
IN LITTERMATE
RAB-
Aqueous humor (50--100 pl) or 12 000 × g supernatant of vitreous humor (100--200 pl) was added to 3 ml of 2,6-dichlorophenolindophenol of known concentration ( a b o u t 0 . 0 4 r a M ) in 5 0 m M p h o s p h a t e b u f fer (pH 6.6) at room temperature (approx. 25°C): mixed, and the decrease in absorbance due to reduction of the dye was measured at 610 nm. From the stoichiometry of the reactions involved, ascorbic acid cone e n t r a t i o n o f a q u e o u s h u m o r a n d v i t r e o u s h u m o r w a s d e t e r m i n e d f r o m c 6 1 0 n m o x i d i z e d d y e = 21 0 0 0 . T e c h n i q u e u s e d f o r e s t i m a t i o n o f a s c o r b i c a c i d o f l e n s is d e s c r i b e d u n d e r M a t e r i a l s a n d M e t h o d s in t h e text. Rabbit
Ascorbic acid concentration Aqueous (mM)
humor
Lens (pmol/g
wet wt.)
Vitreous humor (mM)
Control on regular diet
1.072 + 0.322 (6) *
1.183 ± 0.071
(4)
0.416 ± 0.096 (6)
Experimental on special diet for 2 weeks
0.380 ± 0.080 (6)
0.872 ± 0.071
(6)
0.145 ± 0.071 (6)
Control on regular diet
1.200 ± 0.390 (6)
1.120 ± 0.169 (10)
0.308 ± 0.091 (6)
Experimental on special diet for 4 weeks
0.502 ± 0.214 (6)
0.652 ± 0.078
0.111 ± 0.049 (6)
* Mean ± S.D.; number
(8)
of rabbits studied in parentheses.
0.20 o
o.
,~
O.15
~
O.lO
I1
~
o.05
m
o.0
J
I ~
0.05
O.10
h 0.50
m M H202 F i g . 2. E f f e c t o f H 2 0 2 o n s u p e r o x i d e d i s m u t a s e o f c a l f l e n s in v i t r o . T h e a s s a y s y s t e m h a s b e e n d e s c r i b e d i n c a p t i o n t o T a b l e I. H 2 0 2 ( o e), H202 and 20 mM 3-aminotriazole (X------×) and H202 and 1 mM NaN 3 (o-------o) were added to the assay system, incubated at room temperature (about 25°C) for 10 rain and superoxide dismutase reaction was initiated by pyrogallol. When 3-aminotriazole was used, l e n s e x t r a c t w a s p r e i n c u b a t e d w i t h it f o r 1 h a n d t h e n t a k e n f o r a s s a y . A n a v e r a g e o f 3 - - 4 e s t i m a t i o n s is presented.
35
0.20 .>
o
,_~ 5:
0.15
~"
0.10
\
0.00
t
I
|
I
O.0
0.25
0.50
0.75
I
,,.
1.OO
g Diethyldithiocarbamate injected/kg body wt Fig. 3. E f f e c t o f d i e t h y l d i t h i o c a r b a m a t e on s u p e r o x i d e d i s m u t a s e of r a b b i t lens in vivo. D i e t h y l d i t h i o c a r b a m a t e d i s s o l v e d i n p h y s i o l o g i c a l s a l i n e w a s a d m i n i s t e r e d i n t r a v e n o u s l y t o t h e e x p e r i m e n t a l r a b b i t as a single dose. The c o n t r o l r a b b i t received an i n t r a v e n o u s i n j e c t i o n of e q u a l v o l u m e of p h y s i o l o g i c a l saline. Assay system consisted of 0.2 mM 6-hydroxydopamine, 50 m M K 2 H P O 4 / K H 2 P O 4 b u f f e r (pH 6.5), 0.1 m M N a 2 • E D T A a n d an a p p r o p r i a t e a m o u n t o f e n z y m e e x t r a c t ( p r e p a r e d f r o m l e n s as d e s c r i b e d u n d e r Materials a n d M e t h o d s in the t e x t ) in a final v o l u m e of 1.5 m l at r o o m t e m p e r a t u r e ( a p p r o x . 2 5 ° C ) . O n e unit of superoxide dismutase produced 50% inhibition of the rate of autoxidation of 6-hydroxydopamine as m e a s u r e d b y i n c r e a s e i n a b s o r b a n c e a t 4 9 0 n m . A c t i v i t y ( U n i t s / m g p r o t e i n ) m e a n + S . D . , n = 5 c o n t r o l r a b b i t s a n d a v e r a g e a c t i v i t y o f t h e l e n s e s o f e a c h e x p e r i m e n t a l r a b b i t is p l o t t e d .
66% of control. As estimated, after 2 weeks of feeding the special diet to rabbit, 3-aminotriazole was present in aqueous humor, lens and vitreous h u m o r (Table II). After 4 weeks, the concentration of 3-aminotriazole in eye humors decreased by 20% and in lens by 47%. Table III shows that in rabbit having early cataract, H202 of aqueous h u m o r and vitreous h u m o r was increased by approx. 2--3 times the control. Ascorbic acid in eye humors and lens o f rabbit having early cataract. The data in Table IV show that ascorbic acid of aqueous h u m o r was decreased to approx. 40% of control, vitreous h u m o r to approx. 36% of control, and lens to 58--74% of control, suggesting a fall in the antioxygenic potential of lens. Effect o f H202 on the superoxide dismutase o f calf lens in vitro. Fig. 2 shows that H202 inhibits superoxide dismutase of lens extract. H202 (0.05 mM) caused approx. 8% inhibition which increased to 22--32% in the presence of 20 mM 3-aminotriazole or 1 mM NAN3. A 25% inhibition of superoxide dismutase produced by 0.5 mM H202 was increased to approx. 56% when 3-aminotriazole or NaN3 was added. 20 mM 3-aminotriazole or 1 mM NaN3 produced no effect on lenticular superoxide dismutase. 3-Aminotriazole is a specific inhibitor of catalase [19] and NaN3 is also a p o t e n t inhibitor of this enzyme. Thus, when the protective effect of endogenous catalase of lens was abolished, the inhibitory effect of H2Oz on lens superoxide dismutase was enhanced. Effect o f diethyldithiocarbamate on superoxide dismutase o f lens in vitro and in vivo. Incubation of rabbit lens extract with 1 mM diethyldithiocarbamate produced 40% inhibition of superoxide dismutase activity. Fig. 3 shows that a single dose of 1 g diethyldithiocarbamate/kg b o d y weight in rabbit caused 84% inhibition of lens superoxide dismutase 2.5 h after injection. By reducing the dose to 0.7 g/kg b o d y weight, approx. 50% inhibition was produced after 2.75 h. Further reduction of the dose to 0.5 g/kg b o d y weight
36 resulted in 27% inhibition at 3 h after injection. Our experience with this compound in vivo is not very encouraging because with a dose which causes approx. 50--80% inhibition of superoxide dismutase, the rabbit hardly survives up to 3 h. By reducing the dose to 0.5 g/kg b o d y weight, diethyldithiocarbamate does not cause significant inhibition of enzyme at 3 h after injection. Discussion
Experimental evidence provided here demonstrates the participation of superoxide dismutase and catalase in protecting the crystalline lens from oxidative damage by O2"- and H202. In eye, superoxide dismutase has been previously shown [20] in retinal rod outer segment. We observed that this enzyme is also present in iris, ciliary body, retina, corneal epithelium, corneal endothelium and lens of rabbit and in the lenses of calf and the human. NaCN (1 mM) produced 50% inhibition of superoxide dismutase activity of calf lens extract suggesting that it could be a Cu 2÷- and/or Zn2+-containing enzyme. Further indication a b o u t the Cu 2÷ nature of the lenticular superoxide dismutase was shown b y its inhibition by diethyldithiocarbamate in vitro and in vivo. This c o m p o u n d has been shown [15] to inhibit superoxide dismutase of blood, liver and brain of mice in vivo. H202 has been shown to inactivate (Cu 2÷ + Zn 2÷)-superoxide dismutase [21]. Superoxide dismutase activity of calf lens extract was inhibited by H202 and catalase inhibitor, 3-aminotriazole or NaN3 was found to potentiate this inhibitory effect of H202 by 2--4-fold. In early cataract induced in rabbits b y feeding a diet containing 3-aminotriazole, H20~ concentrations in aqueous h u m o r and vitreous h u m o r were increased by 2--3-fold and superoxide dismutase activity of cataractous lens was decreased. Thus, it is conceivable that in cataract, increased H202 of ocular humors causes inactivation of superoxide dismutase of lens in vivo. The fall in ascorbic acid of aqueous humor, vitreous humor and lens which we observed in early cataract may be due to its oxidation b y O2"-. Oxidation of ascorbic acid by O5"- has been reported [22]. As illustrated in the following scheme (Fig. 4), we propose the enzymatic and nonenzymatic reaction mechanism which may be involved in cataractogenesis. Hydrogen peroxide and molecular oxygen are produced during the disproportionation of the O2'- by superoxide dismutase [12]. Catalase and peroxidase protect the tissues from the toxic effects of H202 [23] and both enzymes are present in eye tissues [8,24]. Selective inhibition of catalase of iris, ciliary b o d y , retina, cornea and lens by 3-aminotriazole resulted in significant increase in H202 of ocular humors of rabbit in vivo. However, under the conditions unaltered GSH peroxidase of eye tissues alone failed to regulate H~O2 in ocular humors within the physiological limit [8]. The increased H202 inactivates superoxide dismutase of lens and possibly of other eye tissues. Thus the enzymatic defense against the toxicity of O5"- being impaired, H202 and 05"could further react, possibly by the Haber-Weiss reaction [25], producing OHwhen H202 concentration exceeds the physiological limit under pathological conditions as in cataract induced by 3-aminotriazole. Besides H202 and O2'- it seems likely that OH-, the most p o t e n t oxidant known, could be the ultimate initiator of cataractogenic process.
37 x
02+
,x Oe"
O~
02: +
2H +
~S,~oo g/ X
H202.1. H202 -t-
Catalase ~2H20 .i. 02
x
Fig. 4. S c h e m a t i c r e p r e s e n t a t i o n of t h e e n z y m a t i c a n d n o n e n z y m a t i c r e a c t i o n s w h i c h m a y b e i n v o l v e d in the m e c h a n i s m of c a t a r a c t o g e n e s i s . SOD, s u p e r o x i d e d i s m u t a s e , AH2, h y d r o g e n d o n o r a n d A, o x i d i z e d d o n o r . D e t a i l e d e x p l a n a t i o n f o l l o w s in t h e t e x t .
To conclude, catalase of eye affords protection to the lens from H202 and it also protects superoxide dismutase of lens from the inhibitory effect of H202. Superoxide dismutase, in turn, protects the lens from 02"- and it is possible that b o t h superoxide dismutase and catalase might prevent the formation of
OH.. Acknowledgements This investigation was supported by NIH research grant No. EY01731 from the National Eye Institute, Department of Health, Education and Welfare, United States Public Health Service. Our sincere thanks are due to Dr. Harold L. Kern for going through the manuscript and for his helpful suggestions and Dr. Arnold I. Turtz, Dr. R. David Sudarsky and Dr. Herbert M. Katzin for their generous encouragement. We dedicate this paper to Professor L.P. Agarwal and Professor R.K. Mishra.
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