Exp. Eye Res. (1992) 55. 671-678

Biochemical Changes and Cataract Formation in Lenses from Rats Receiving Multiple, Low Doses of Sodium Selenite LI-LI

HUANG”“,

CHANG-YING

ZHANG”,

JOHN

L. HESSbfAND

G. E. BUNCEb

a Department of Biochemistry, Beijing Medical University, Beijing, P.R. China, and b Department Biochemistry and Nutrition, Virginia Polytechnic Institute and State University, Blacksburg, VA 25061, U.S.A. (Received

Norwich

6 November

1991 and accepted

in revised

form 28 February

of

1992)

Nuclear cataract formed in rat lens in response to a protocol of multiple, low dosesof sodium selenite. Nuclear cataract occurred, in both Wistar and Sprague-Dawley rats. following five subcutaneous injections of selenite over an S-day period with an accumulated dose of 40-50 nmol selenite go1 body weight. Glutathione content decreased within the first 24 hr of treatment and remained at 600/, of controls. Lipid peroxidation occurred in Wistar rats prior to nuclear cataract formation. A two to threefold increase in calcium concentration and decreased protein content accompanied nuclear cataract development. Enzyme activities were measured for glutathione peroxidase, glutathione reductase. and glutathione S-transferase, and only the peroxidase activity remained constant through the period of cataract formation. This protocol resulted in nuclear cataracts similar in appearance to those observed with a single. acute dose of selenite. The opportunity to control the rate of selenite-dependent formation allows further definition of precataractous events. Kq words: lens : cataract ; selenite: oxidation ; calcium: glutathione peroxidase.

1. Introduction Since the initial observation of nuclear cataract in rats treated with doses of sodium selenite (Ostadalova, Babicky and Obenberger, 1978), much information about this cataract has been reported (Bhuyan, Bhuyan and Podos, 198 1; Bunce and Hess, 1981; Shearer, David and Anderson, 1987). Other studies of selenite effects on lens include work with in vitro exposures of cultured rabbit lenses with specific evaluation of effects on the epithelium (Hightower and McCready , 199 1). Accumulation of calcium accompanies a loss of protein, and nuclear cataract development occurs in the absence of significant lens swelling. In contrast, opacification in the ‘sugar’ cataract is accompanied by a 10% decrease in protein but significant swelling as a result of a three-fold increase in lens water content (Sipple, 1966). The selenite-induced cataract model has advantages in its reproducible occurrence and short time span (4-5 days) required for nuclear cataract development. Our objective was to establish a protocol for a slower, more controlled rate of cataract formation in response to a selenite stress. Such a result would enhance the study of factors that might prevent cataract formation. Wistar rats were used at Beijing Medical University to describe a protocol for observing a reproducible nuclear cataract following five injections of selenite (Huang, I 989 1. We further describe results of experi* Current address: Human Nutrition Research Center. Tufts liniversity. 71 1 Washington Street. Boston, MA 02111. U.S.A. t For reprmt requests at: Department of Biochemistry and Nutrition, 207 Engel Hall. Virginia Polytechnic Institute and State IJniversity. Blacksburg. VA 24061, U.S.A.

001+4835/92/l

10671 fO8 $08.00/O

cataract

merits with Sprague-Dawley rats, animals on which most of the characterization of the selenite-induced cataract has been reported (Shearer et al., 1987). Reproducible cataract formation occurred after 10 days of treating animals with multiple, low doses of sodium selenite. This interval is 2.5 times longer than that required for nuclear cataract formation in the response to a single acute dose of selenite (Ostadalova et al., 1978).

2. Materials and Methods Animals and Tissue Animals were maintained under conditions of 6 5 % relative humidity, 25 “C and a day/night cycle of 12 hr. All procedures involving animals conformed with the ‘Guide for the Care and Use of Laboratory Animals’ (DHEW, NIH 86-23). Litters were housed in individual cages with both the male and female parent through weaning, and were provided with laboratory chow and water ad libitum. Animals were given subcutaneous injections of 1 mM Na,SeO, in 0.9% NaCl in the scruff of the neck. For the optimum protocol in Wistar rats, the first injection was 6 nmol Na,SeO, g-’ body weight on day 13. Each subsequent dose of selenite was increased by 1 nmol g-’ body weight administered every other day for a total of five injections. Wistar rats were scored for cataract after the eyes were dilated with 1 y0 atropine and examined with a slit lamp. Excised lenses from Sprague-Dawley rats were carefully examined with bright and dark field illumination using an Olympus dissecting microscope 0 1992 Academic Press Limited

L-L. HUANG

672

equipped with a below stage illuminator (Model SZHILLD, Olympus. Japan). Lenses were removed immediately after decapitation of the animal. Careful removal of vitreous and attachments preceded analysis. Lenses were weighed and homogenized as indicated below or were washed with 100 ill 0.5 mM EGTA in 0.9 y0 saline, blotted on EGTA-soaked paper, and dried at 110°C to constant weight. Glutathione Analysis Glutathione was analysed in 5 % trichloroacetic acid or 0.9 M perchloric acid extracts following homogenization and centrifugation at 4°C using the assay for nonprotein sulkydryl (Sedlak and Lindsay, 1968), or the cycling assay (Brehe and Burch, 1976). Glutathione and DTNB [5,5-dithio-bis-(2-nitrobenzoic acid)] were from Roth Chemical (Germany) or Sigma (St Louis, MO. U.S.A.). Lipid Peroxide Analysis Lipid peroxides were quantified with the thiobarbituric acid (TBA) reaction (Yagi, 1976 ; Ohkawa, Ohishi and Yagi, 1979). Homogenates of four lenses were prepared in a ratio of 1 g 10 ml-’ of 1.15 % KC1 in a glass homogenizer. The homogenate was made 0.4% (w/v) SDS and 7.5% (by volume) acetic acid, 0.3 % TBA at pH 3.5. This solution was diluted 1:2 with water and heated at 95” for 1 hr. After cooling the reaction product was extracted with a solution of n-butanol : pyridine (15 : 1, by volume). Fluorescence at 553 nm of the organic layer was measured with an excitation at 5 15 nm ; 1 , 1,3,3-tetramethyoxypropane (TMP) was used as a standard. TBA was obtained from BDH Chemical Ltd. (U.K.) and TMP was from Sigma. Enzyme Assays Samples were prepared for protein and enzyme analysis for enzymes associated with glutathione metabolism in the lens. A 10% (w/v) homogenate of lens was prepared in 50 mM sodium phosphate, pH 7.0, containing 3 mM EDTA. After centrifugation at 15 000 g for 15 min the supernatant was assayed directly for protein (Bradford. 1976) and enzyme activities. Yeast glutathione reductase was obtained from Boehringer or Sigma. Reduced (GSH) and oxidized (GSSG) glutathione. NADPH, l-chloro-2,4dinitrobenzene (CDNB), and bovine serum albumin (for protein standard) were also Sigma products. Glutathione peroxidase. Activity was monitored at 340 nm at 25’C according to the method of Paglia and Valentine (1967), but modified to have the following final concentrations: 10 mM GSH, 0.3 mM NADPH, 1 mM NaN,, 50 mM sodium phosphate, pH 7.0. 3 mM EDTA, and 0.6 units of yeast glutathione reductase. After addition of sample, the reaction was

ET AL

initiated with sufficient H,O, to a final concentration of 0.03 mM. Glutathioue reductase. Activity was determined by the glutathione-dependent oxidation of NADPH at 340 nm at 30°C (Carlberg and Mannervik. 19 7 5 ). The final concentrations in the reaction were 0.1 rnM NADPH, 1 mM GSSG. 0.5 rnM EDTA. 100 rnbt phosphate at pH 7.6. GZutathione S-transferase. Activity was determined by measuring the rate of CDNB conversion at 340 nm (Habig, Pabst and Jakoby. 1974). The reaction contained 1 mM CDNB, I mM GSH, 100 mM sodium phosphate, pH 6.5 and sample. The absorptivity for CDNB is 9.6 x 10” cm-’ M-‘. Elemental Analysis Selenium. All reagents were obtained at purest quality and water was distilled and deionized. Diaminonaphthalene dihydrochloride (DAN) (Fluka, Germany) was the fluorimetric reagent (Lalonde et al., 1982). To six lenses, 500 /rl HNO, and 500 ~1 HClO, were added and the mixture was slowly heated ( 1 hr) at 13 5“C. The temperature was increased to 185°C for 2 hr. A solution of 10 /lg cresol red mll’ of 20 mM EDTA was added to the cooled tubes and the pH was adjusted to l-2 with NH,OH. The final volume was adjusted to 10 ml after the addition of 5 ml 100 mM HCI. Under subdued illumination, 0.5 ml DAN ( 1 g I-’ 100 mM HCl) was added and the solution incubated at 5 5°C for 20 min. The piazselenol complex was extracted into 3 ml cyclohexane and the fluorescence of the organic phase was measured at 520 nm with an excitation beam at 360 nm. Calcium. Dry lenses were digested alternately with nitric acid and hydrogen peroxide through two cycles. Total Ca in lens was measured by means of atomic absorption spectrophotometry at 412 nm (Bunce, Hess and Batra, 1984). Statistics All values are reported as the mean+s.a. Comparisons were evaluated with Student’s t-test and significant differences determined when P < 0.01.

3. Results incidence of Cataract In Table I we report experiments with Wistar rats for different initial doses of selenite followed by four subsequent doses each increased by a constant amount of 1 nmol g-’ body weight (bw). If the initial dose was less than 6 nmol g-’ bw, not ail lenses formed cataract. At total doses in excess of 50 nmol g-’ bw both the cortex and nucleus of the lens became opaque rather than the nucleus alone. In a second experimental series (Table II) each group of Wistar rats was given a constant initial dose

CHRONIC

SELENITE

CATARACT TABLE

673

I

TABLE II

Response of rat lens to initial and accumulated dose of sodium seknite

Group A B

G I K

Initial Se dose (nmol g-I bw)

Accumulated Se dose (nmol 8-l bw)

Cataract* (%I

4 5 6 7 8

30 35 40 45 50

0 60 100 100 100

Required increase in selenite dose to cause nuclear cataract formation

Group C D G Sprague-

Increased Se dose (nmol g-’ bw)

Accumulated Se dose (nmol g-’ bw)

Cataract* (“/,I

30 35 40 50

0 10 100 100

0 0.5 1 2

Dawleyt

Thirteen-day-old Wistar rats were given initial doses as indicated, and subsequent doses were increased by 1 nmol g-r bw at each injection every other day. A total of five injections was administered. * Cataract was evaluated at day 22. All cataracts were nuclear opacities with the exception of those in group K: these cataracts had both an opaque cortex and nucleus. At least ten animals were evaluated for each experiment.

of 6 nmol g-’ bw and a range of subsequent increments was evaluated. When the incremental increase was less than 1 nmol g-’ bw cataract incidence was less than 100%. At accumulated doses of less than 40 nmol Se g-’ bw no additional cataracts formed after day 22, 9 days after the initial dose of selenite. Lenses were observed through 50 days of age. For the Sprague-Dawley rat, 100% incidence of cataract occurred only when the total dose was 50 nmol. The optimum protocol for development of cataract

Thirteen-day-old Wistar rats were given an initial dose of 6 nmol g-r bw. and subsequent doses were increased as indicated at each injection every other day. Five injections were administered. * Cataract was evaluated at day 22. All cataracts were nuclear opacities. At least ten animals were evaluated for each experiment. t Sprague-Dawley rats required an accumulated dose of 50 nmol Se g-r bw. and 100% cataract was observed only when animals were 12 days old or younger at time of initial injection.

in this model was based on an initial dose of 6 nmol Se g-’ bw. Each subsequent dose was increased by 1 nmol Se g-’ bw. In Wistar rats the first injection may be given as late as 13 days of age with subsequent injections on days 15, 17, 19, and 2 1. The total number of five injections resulted in a gradual increase in selenite dose to a final total dose of 40 nmol g-’ bw. After three injections of selenite, slight opacities appeared in the lens of Wistar rats as

FIG. 1. Appearance of lenses during the course of cataract formation in lenses from rats treated with multiple low doses of sodium selenite. Age-matched lenses from control animals (C and E) are compared with lenses from 18-day-old Sprague-Dawley rats

which

had received

three

injections

of sodium

selenite

beginning

at age 13 days

(D);

the accumulated

dose was

2 1 nmol g-’ bw. The mature nuclear opacity appears in the 22-d-old lens after the fifth injection in the rat (F): the accumulated dose was 50 nmol g-l bw.

674

L.-L.

TABLE III

HUANG

ET AL.

TABLE IV

Changes in lens weight j&wing

selenite treatment

Protein content in lenses Jrom control and selenitetreated rats

Lens weight (mg per lens) Rat age (days)

-.~ Control (n) _-~~.

Wistar 18 22 Sprague-Dawley 22 2 1 (acute model)

Protein content (mg protein rng-’ lens) ~~~- ~~ ~~ ~~~ Control Selenite

Selenite (n) -~ -.

16.6kO.9 18.8f1.4

(12) (13)

15.6f1.5 16.9*0,7*

(18) (15)

17.9kO.9 18.0+0.5

(10) (12)

14.5*0.4* 15.0+1.7*

(10) (17)

Animals received three doses of selenite by day 18, and lenses showed central opacities in the nucleus. By day 22. after five doses of selenite, all lenses showed opacities involving the whole nucleus with little cortical opacity. Values are means + S.E. * Selenite group was significantly less than control. P < 0.0 1.

Chronic Wistar Total protein Soluble protein

Sprague-Dawley Soluble protein Acute Sprague-Dawleyl Soluble protein

039*001

0.3 5 + ow

t1~23+0~01

ct.19 + O-02”

U-28 + 0.02

0.20 * 0.0 1%

0.31 _$O.Ol

0~30~0~01

Lenses were from 24.day-old animals (the final dose of selenite was given on day 20 or 11). All lenses from treated animals had mature nuclear cataract with a generally clear cortical region. Values are mean + sx. * Selenite group was significantly less than control. P < O,Ul. n = 11. t Lenses were from 1h-day-old rats. 4 days after a single dose of selenite.

vacuoles in the lens cortex beneath the epithelium, but the nucleus remained clear. Nuclear cataract was observed 24 hr after the fifth injection of selenite. Mature nuclear cataract occurred in all lenses of Wistar rats by this stage of treatment, days 22-23. When injections were begun not later than at day 12 in Sprague-Dawley rats, nuclear cataract appeared in all animals using an initial dose of 6 nmol Se g-’ bw and subsequent doses increased by 2 nmol g-l, the accumulated dose was 50 nmol g-’ bw (Fig. 1). Microscopic examination of Sprague-Dawley lenses revealed neither obvious morphological changes nor opacities 24 hr after the initial injection of selenite.

standard protocol that delivered 40 nmol g-’ bw to the Wistar rat and 50 nmol g-’ bw to the SpragueDawley rat. A trend to lower lens weight was observed after three doses of selenite were administered. A significant decrease in lens protein was also observed in lenses with cataract (Table IV).

Lens Weight and Protein Content

Level of Non-protein (Glutathione) Suljhydryl Groups

As indicated in Table III the lens weight was significantly less in all the treated animals using the

Over 90 % of nonprotein sulfhydryl groups in lenses is glutathione. A significant decrease in glutathione

7

T

Sprague-Dawley rot < I-

6t

Accumulated

selenlte

dose (nmol 4’ body weight)

FIG. 2. Glutathione content of lenses from Wistar and Sprague-Dawley rats following multiple, low doses of sodium selenite. The protocol for administering the selenite begins at 6 nmol g-l bw to 13-day-old rats. Subsequent doses, administered on odd days of age, were increased by 1 nmol g-’ bw for Wistar rats and by 2 nmol g-’ bw for Sprague-Dawley rats. Data from Sprague-Dawley rats are given only for lenses receiving the total number of injections. Values are estimated from determinations on at least ten lenses which were removed from the animal 24 hr after the last injection. SE. was less than 5% of the mean. For all controls (m) glutathione content was significantly greater than that occurring in lenses from treated animals (@), P < 001. Only lenses from the 22-day-old treated animals had mature nuclear cataracts.

CHRONIC

SELENITE

CATARACT

675

TABLE V

level was observed in lenses from selenite-treated Wistar rats (Fig. 2). One day following the initial injection of selenite, lens glutathione decreased to 66 % of control levels. This concentration remained at 54% and 62 % of normal after the third and fifth injections of selenite. A similar decrease also characterized glutathione content in the Sprague-Dawley lens after five injections of selenite.

Selenium content of lenses from Wistar rats

Lens content* Se dose (nmol g-’ bw) (Se, ng per lens)

-.~ ~~ Chronic model 18 day, control None Three injections 21 (chronic) 22 day, control None Five injections 40 (chronic) Acute model (Bunce and Hess, 1981) Sprague-Dawley rats 12 day, control None One injection (acute) 30

1.450.28 3.5 f0.27 2.OkO.29 3.4 * 0.30

Selenium

2.4 f 0.24 7.9 k 0.65

As shown in Table V, the selenium content of lenses from selenite-treated rats was significantly increased over controls. The level remained at approximately 3.5 ng Se per lens during the final period of the treatment, but this concentration was less than the amount of Se (7.9 ng Se per lens) present in the acute model at 24 hr post-injection (Bunce and Hess, 198 1).

Lenses were removed from animals 24 hr following the last injection of selenite. Values are mean f S.E. * All treatments are significantly different from controls, P < O%)l. For each determination, n > 6.

Lipid Peroxidation TABLE

As shown in Table VI, a significant increase in malondialdehyde (MDA) occurred prior to and accompanying nuclear cataract formation. MDA concentration in lenses from Wistar rats receiving three injections of selenite increased by 90% over control. This increase was sustained through the fifth injection and the appearance of nuclear cataract.

VI

Lipid peroxidation in lenses from Wistar rats Malondialdehyde content (pmol MDA g-’ lens) Animal age ~Selenite Control (days) -~~ ~~~~~..~ 206 & 41.8* 18 llOk3.4 22 114kl1.6 202*27.3*

~

Enzyme Activity

Eighteen day-old rats received three injections of selenite (2 1 nmol g1 bw): 22-day old rats received five injections of selenite (40 nmol g’ bw). Values are the mean f S.E. of at least six samples. * Selenite group was significantly different from control group. P < 0.01.

TABLE

Data in Table VII show activities of key enzymes catalyzing glutathione-dependent reactions in the lens. Glutathione peroxidase activity increased by 2 5 % in the early stage of cataract development only in Wistar

VII

Enzyme activities in lenses from control and selenite-treated rats Enzyme activity (units mg-’ lens) ~~-GSSG reductase GSH-S-transferase GSH peroxidase

--~~-

_.~~~ -

Treatment Chronic model Wistar Control (18 day) Three injections Se Control (22 day) Five injections Se Sprague-Dawley

Control, (16 day) Five injections Se Acute model One injection Set

2.5kO.36 3.2 f 0.45* 2.2 f0.16 2.3 k 0.30

0~20+0~01 0.18 kO.01 0.19 kO.02 0.13+0.02*

0.82 4 0.03 0.5940.10* 0.8340.10 0.60 t- 049*

2.6+0.15 2.5 kO.25

0.8 1-t 0.046 0,28*0.035*

1.01 + 0.08 030 4 0.08*

2.5 kO.12

0,56f0.075*

1.20&O-5

Enzyme activities were measured in extracts from lenses removed from animals 24 hr after the last injection of selenite. Cumulative doses of selenite were 21 and 40 nmol g-l bw for the 18 and 22-day-old animals or 30 nmol g-’ bw for the acute dose. All lenses, except for the lenses from l&day-old treated animals, had mature nuclear cataract. Values are mean+S.E. of at least six samples. * Treatments were significantly different from controls. P < 0.01. All values were estimated from n > 6. t Lenses from

1 A-day-old

rats. 4 days after treatment

with

a single,

acute dose of selenite.

L.-L. HUANG

676 TABLE VIII

Calcium content in cataractous lens from Sprague-Dawley rats treated with either the chronic or acute protocol Calcium content (jtmol Ca g--l dry wt) Model Chronic Acute

Control 0.64kO.22 0.6650.22

Se-treated 6.6_+0,52 5.0 kO.23

Animals were 12 days old at the initiation selenite treatment (chronic model) for 15 days old (acute model). Lenses were analysed for Ca content when the animals were 23 days old (chronic model) or 20 days old (acute model). The age-matched control lenses were clear and lenses from the selenite-treated rats had mature nuclear cataract. Data are representative of many experiments but these means were based on n= 3.

rats. Glutathione reductase and glutathione-s-transferase activities decreased 15 % and 30 %, respectively, along with the occurrence of nuclear cataract. These decreases were associated with decreased protein in these lenses, and in the Wistar rat with the extent of cataract severity. Hence, the initial increase in glutathione peroxidase activity in response to selenite treatment did not persist during maturation of the nuclear cataract while the reductase and transferase activities became lower than controls. In the experiments with the Sprague-Dawley rats, similar results were observed. The peroxidase appeared to be unchanged in lenses with nuclear cataract with either the chronic or acute delivery of selenite. Significantly lower activities accompanied nuclear cataract formation for both the reductase and transferase during the chronic delivery of selenite ; however, only the reductase activity seemed to be less when evaluated in the lenses with nuclear cataract from animals receiving the acute dose of selenite (Table VII).

Calcium The data for Ca analyses appear in Table VIII. The mature cataract in the chronic model is accompanied by an increase in Ca content of a magnitude similar to that observed in the acute model (Bunce et al.. 1984).

4. Discussion Over the past 10 years we have used the seleniteinduced cataract model to quantify and characterize events that occur in the rat lens prior to the formation of nuclear cataract. Oxidative stress might explain some of the responses that have been described in the lens from selenite-treated rats (Shearer et al., 1987). However, our repeated attempts to prevent the onset of the cataract with antioxidants have not been successful when a single dose of 30 nmol

ET AL.

selenite g-l bw is used. Only recently was there a report that mega doses of vitamin C. administered as IP injections, could reduce the incidence of seleniteinduced cataract (Devamanoharan et al.. 199 I 1. It was our goal, therefore, to develop a protocol for inducing nuclear cataract at a slower, yet reproducible rate, in the rat lens. A more controlled rate of cataract formation could provide greater opportunity for interrupting the sequence of events that leads to nuclear cataract and, thus. prevent cataract formation. If well defined, such a model would allow careful evaluation in vivo of treatments and agents for protecting the lens. With the protocol described in this report, we successfully produce, by using multiple low doses of selenite, a nuclear cataract that develops over a loday period in the rat lens. We designate this model as the chronic model. In contrast, the well documented acute model requires a single dose of selenite introduced within a specific age at time of treatment and results in mature nuclear cataract four days after administering the selenite dose (Ostadalova et al., 1978). In the chronic model, the initial dose of Se (Table I), the total dose of Se (Table II) and the age of the animal are critical parameters (Barbicky et al.. 1985). The available, direct blood supply to the lens decreases during the first 14 days of post-natal development in the rat (Latker and Kuwabara, 1981). and the lens itself is undergoing specific developmental changes (Groth-Vasselli and Farnsworth, 1986. 1987). Hence, the delivery of selenite to the lens may depend on critical developmental events. In order to observe highly reproducible cataract formation (Fig. 1) in all lenses in the chronic model, animals must be no older than 13 days for the Wistar rat or 12 days for the Sprague-Dawley rat. A minimum initial dose of selenite of 6 nmol g--l bw is required for cataract formation. If the final dose of selenite. normally given at day 21 is not provided, nuclear cataract will not develop. Hence. the final delivery of selenite in the chronic model is at a time when selenite is without effect when given as an initial dose in the acute model. Additional information about biochemical and developmental changes during cataract formation are required in order to understand these properties of the chronic model. One characteristic of the acute, selenite-induced cataract is the decrease in glutathione content in the lens which precedes nuclear cataract formation (Bunce and Hess, 1981). Within the initial 24 hr of the single injection of selenite, the glutathione concentration decreases to 40% of that observed in agematched controls. However, the concentration gradually increases so that, at the time nuclear opacity is observed, the glutathione content has returned to 80% of control. In the present chronic model, glutathione content also decreases in the lenses from selenite-treated rats (Fig. 2). The continuous delivery

CHRONIC

SELENITE

677

CATARACT

of selenite results in a generally lower (40-50x) content of lens glutathione at the time of nuclear opacification. Perhaps the selenium content of the lens must return to normal before the glutathione content can be restored. Cataract also forms as a consequence of glutathione depletion, due to swelling of lens fiber cells in the young mouse lens (Calvin et al., 1991) and in galactose-induced ‘ sugar ’ cataract (Supple, 1966 1. The Se in the lens (Table V) of the chronic model increases but not as much as the Se content reported for the acute model (Bunce and Hess, 1981). On the other hand, the level of selenite remains at 3’5 ng per lens through the actual appearance of nuclear cataract. This result is consistent with the sustained delivery of the lower dose of selenite used in these experiments. The prompt onset of mature nuclear cataract requires the final dose of selenite. How selenite triggers the loss of Ca-homeostasis is not fully understood, but the appearance of the lens and its Ca content at day 22 (Table VIII) are consistent with the activation of calpain as the step which precipitates nuclear cataract in both models (David and Shearer, 1984). Further experiments are needed to establish the relationship between selenite dose and the changes in Ca content. A decrease in lens weight and lens soluble protein (Tables IV and V) accompanies the progress of cataract formation in the chronic model as is the case with the acute model (Bunce and Hess, 198 1). These changes that accompany formation of the nuclear opacity are consistent with the occurrence of extensive proteolysis observed in the acute model as established by David and Shearer (1984). A marked increase occurs in lens MDA particularly in the Wistar rat (Table VI). This response agrees with the increase reported for Sprague-Dawley rats by Devamanoharan et al. (19911, although the absolute concentration in those animals is 50 times greater than the quantity we report here for the lenses from Wistar rats. Further, in the severe cataracts observed by Bhuyan et al. (198 1). elevated lipid peroxidation was reported following multiple injections of selenite into rats. A loss of enzyme activities was associated with the onset of cataract (Table VII). Decreases were noted in glutathione reductase and glutathione-S-transferase activities. Glutathione peroxidase activity. however, was maintained even in the presence of a decrease in soluble protein. This result might reflect a different distribution of peroxidase in the lens compared to that for the reductase and the transferase. The major loss of soluble protein in lenses from selenite-treated rats was consistent with the lower lens weight that is attributed to proteolysis in the nucleus. Hence, if the peroxidase was associated with the epithelial cells, its activity could be less affected than would be proteins more uniformly distributed in the lens. Conservation of peroxidase activity might explain the greater stabiIity of the cortical region and ability of the lens to grow

following the initial insult of selenite. Further quantitative studies, however, are required to establish distributions of enzyme activities between the cortical fiber cells and the epithelial cells of the young rat lens. The description of a more slowly developing cataract in response to selenite stress provides a longer opportunity to examine metabolic events which contribute to this pathology. Also, the reproducibility which results from the described protocols for Wistar or Sprague-Dawley rats establishes a basis for evaluating agents that may protect against selenite stress (Huang et al., 1989). The acute and chronic models provide opportunity to describe and to quantify effects of oxidative stress through systemic delivery of selenite to the lens. Such information is valuable for comparing and interpreting the responses by cells or tissues to in vitro exposures of oxidative stress.

Acknowledgements The authors appreciate the excellent technical contributions of Wei-Hong Jia (Beijing Medical University) and Alice Tira (Virginia Polytechnic Institute and State University) and the support of the National Institutes of Health, NE1(ROl EY06123).

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