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Analysis of Glucose Levels During Glucocorticoidinduced Cataract Formation in Chick Embryos Hideo Nishigori, Jung W. Lee, Yasuhisa Yamauchi, Kozuo Maruyama, and Mofoharu Iwatsuru When 15-day-old developing chick embryos were administered hydrocortisone hemisuccinate sodium (HC; 0.25 fimol/egg), the content of glucose in the lens markedly increased from around 6 hr, and reached about 25-30-fold above the matched control at 24-48 hr. Thereafter, the glucose level declined and returned to the control level by 100 hr. The profile of lenticular glucose levels was similar to that of the appearance and disappearance of lens opacification. Prednisolne, as well as HC, produced cataract and the elevation of glucose in the lenses. Cortexolone and cortisone, which have weak or negligible glucocorticoid activity in developing chick embryo, could neither produce cataract nor the elevation of glucose in the lenses. An attempt was made to find similarity between this glucocorticoid-induced cataract and sugar cataract known in mammals. In both control and HC-induced cataract (stage IV-V) obtained 48 hr after HC administration, sorbitol, fructose, and glycosylation of protein could not be detected. Dehydration was observed in HC-induced cataractous lens. These data demonstrate that the glycosylation of lenticular protein and the accumulation of polyol were not involved in glucocorticoid-induced cataract formation in developing chick embryos. These results suggest a relationship between the elevation of glucose and cataract formation. However, when cataract formation was blocked by ascorbic acid treatment, the glucose level remained high. Therefore, any relationship between glucose level and cataract may be complex or indirect. Invest Ophthalmol Vis Sci 28:168-174, 1987 is maintained at a level about 2.5 mg/ml, 8 or by inducing diabetes with drugs.9 The increase in lens glucose in cataract is suggested to be caused by the increase membrane permeability. In the present paper, we show that the glucose level in the lens increased markedly with lens opacification and decreased with recovery from cataract after hydrocortisone administration. This relationship between the alteration of glucose level and hydrocortisone-induced cataract formation is discussed in regard to the hypotheses on the formation of sugar cataract in mammals. 9 ' 11 Previous studies have shown that treatment with hydrocortisone, an adrenocortical hormone, can produce reversible cataract in developing chick embryos. The level of glutathione 12 and ascorbic acid3 in the lenses decreased with cataract formation after hydrocortisone administration, but returned to the control level with recovery from cataract. This hydrocortisone-induced cataract formation was effectively prevented by the administration of radical scavengers, certain SH-compounds, 2 and ascorbic acid.3 These results suggest that cataract formation by hydrocortisone may be caused by oxidative attacks produced in ovo. However, since hydrocortisone is a typical glucocorticoid hormone which causes the alteration of metabolic activities in various tissues,4'5 several other possibilities should be considered for the mechanism of cataract formation by hydrocortisone. We have focused on glucocorticoid-produced hyperglycemia in mammals,4 since cataracts are found in a variety of diabetic animals. For instance, in young rats, sugar cataracts have been produced by directly feeding galactose or xylose,6'7 or by a hyperglycemia where the blood glucose Materials and Methods One-day-old fertile White Leghorn eggs were purchased from a local hatchery and incubated in a humidified incubator at 37.5°C. To 15-day-old embryos, hydrocortisone hemisuccinate sodium (HC; 0.25 /xmol in 0.2 ml sterilized water) as a glucocorticoid was administered through a small hole in the eggshell on the air sack. '"3 This single dose was based on dosage studies described previously.1 At the indicated time after HC administration, the lens and the veinous blood were obtained from the embryos. Prednisolone hemisuccinate sodium (PS), cortisone hemisuccinate sodium (CS), and cortexolone hemisuccinate sodium (CX) were also administered in the same way. From the Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko Kanagawa, Japan. Submitted for publication: December 3, 1985. Reprint requests: Dr. H. Nishigori, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa, Japan 199-01. 168 Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933362/ on 05/07/2017 No. 1 169 ELEVATION OF GLUCOSE IN GLUCOCOPJICOID CATARACT / Nishigori er ol. Determination of Glucose Determination of Sorbitol Twenty lenses were sonicated in 0.5 ml of distilled water and the volume adjusted to 0.7 ml with water. To this homogenate, 0.7 ml of ice-cold 2 N perchloric acid was added to deproteinize. After neutralization with 2 N potassium hydroxide, a centrifugation was carried out. Whole supernatant was used for the assay described by Bergmeyer et al.13 Determination of Fructose Twenty lenses were sonicated in 0.5 ml of distilled water and the volume adjusted to 0.7 ml with water. Four percent perchloric acid (0.3 ml) was added to this. After a centrifugation, the supernatant was assayed as described by Bernt and Bergmeyer.14 Determination of Glycosylation of Protein Ten lenses were sonicated in 1 ml of distilled water. The sample was dialyzed against 100 ml of water to remove free glucose, and was then subjected to the colorimetric test of Fluckiger and Winterhalter by using thiobarbituric acid.15 5-Hydroxymethylfurfural was used as standard. Determination of Protein Protein in lens was determined by the method of Lowry et al.16 Determination of Glutathione Glutathione in lens was determined with the use of Ellman's reagent.17 Hydrocortisone hemisuccinate sodium, prednisolone hemisuccinate sodium, cortisone, and ascorbic acid were obtained from Sigma Chemical Co. (St. o • Cont. : HC : 20 moL E m \ 3io ucose Fifteen lenses were homogenized in 0.7 ml of double distilled water by a sonicator (Branson Sonic Power Company, Connecticut) and mixed with 0.3 ml of 4% perchloric acid. After a centrifugation, 0.1 ml of the supernatant was used for the determination. Glucose was determined by an enzymatic method using a kit of Gluco-Quant (Boehringer Mannheim GmbH, Mannheim, W. Germany) based on the method described by Bergmeyer et al.12 The amount determined by this kit shows the sum of glucose and glucosesphosphate. When the actual glucose ("glucose") amount was required, glucose-6-phosphate, determined by the method described by Bergmeyer et al,12 was subtracted. The method of expression, nmol/lens, closely relates to tissue concentration, since variations in lens wet weight were small. &* o r\ i i 03 10 i • 2024 i 48 T i me ( hr l 72 l 96 Fig. 1. Blood glucose level in developing chick embryos after HC administration. HC was administered to 15-day-old chick embryos (0 hr). Blood was collected at the indicated times after HC administration. Glucose content was determined as described in Materials and Methods. Data were expressed as mean ± SE (n = 5-30). In the cases of circles without bars, the bar ranges were smaller than the diameters. Louis, MO). Cortisone hemisuccinate sodium and cortexolone hemisuccinate sodium were prepared from cortisone and cortexolone (Aldrich Chemical Co., Milwaukee, WI).18 Glucose-6-phosphate dehydrogenase, phosphoglucose isomerase, sorbitol dehydrogenase, ATP-disodium, NADP, and NAD were obtained from Boehringer Mannheim Yamanouchi Co., Tokyo. Other chemicals were of analytical grade. Results When 15-day-old chick embryos were administered a single dose of HC, an opaque ring appeared between the cortical and the nuclear regions in their lenses during the first 24 hr, and the nuclear region became opaque by 48 hr with a high incidence. By 96 hr after treatment, the opacity had disappeared.1 During this treatment period, the blood glucose level was found to change, increasing to 1.5-2-fold above control between 20 and 72 hr, but returning to control level at 96 hr (Fig. 1). The elevation of blood glucose was demonstrated to be dependent on the dose of HC. The administration of sodium succinate (0.25 /imol/egg) which was easily released from HC in ovo only slightly elevated blood glucose (Table 1). Blood glucosesphosphate could not be detected in either control or HC-treated animals (not shown). It has previously been demonstrated that an elevation of blood glucose level induces an increase of glucose in the eye, including the lens of mammals.6"8 Figure 2 shows the profile of glucose levels in the lens after HC administration. During normal growth of chick embryos from 15 (0 hr) to 19 (100 hr) days old, the glucose in the lens remained unchanged at approxi- Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933362/ on 05/07/2017 170 Vol. 28 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / January 1987 Table 1. Effect of different amounts of HC on blood glucose of developing chick embryos %of Dosage nmole/egg mmole/l 7.45 ± 0.43 (33) 100 HC 0.25 0.025 0.0025 15.80 ±0.74 8.57 ±0.51 7.94 ±0.09 (35) (7) (7) 212 115 107 Sodium succinate 0.25 8.60 ±0.22 (7) 115 Control Number of embryos Table 2. The level of glucose-6-phosphate in control and HC-induced cataractous lens control HC at the indicated dosage and 0.25 jimole of sodium succinate were administered to 15-day-old chick embryos, and 48 hr later their blood was collected. Glucose was determined as described in Materials and Methods. Data were expressed as mean ± S.E. mately 2 /umol/lens. When chick embryos were given HC on day 15, however, the level of glucose in the lens began to increase prior to the appearance of an opaque ring; the glucose in the lens began to increase at around 6 hr of HC administration, and reached 25-30-fold above the matched control between 24 and 48 hr. Interestingly, its level returned to the control level by 100 hr after HC treatment with recovery from cataract. Glucose in the lenses removed at 48 hr after HC administration was determined separately as "glucose" and glucose-6-phosphate (Table 2). The amount of Control Glucose-6-phosphate "Glucose" Glucose-6-phosphate HC-treated Fold increase 1.95 ±0.53 (4) 2.73 ±0.76 (4) 1.57 ±0.26 (4) 44.34 ±0.13 (4) 1.24 "glucose" 1.4 28.2 0.06 Determination of glucose-6-phosphate: 0.7 ml of twelve lenses sonicated in water was mixed with 0.3 ml of 4% perchloric acid. After a centrifugation, 0.1 ml of the supernatant was used for assay described by Bergmeyer et al.12 nmol/ lens was calculated. Four experiments were carried out and data were expressed as mean ± S.E. "glucose" (1.95 ± 0.53 nmol/lens) and glucosesphosphate (1.57 ± 0.26 nmol/lens) in control lenses were similar. However, the amount of glucose-6-phosphate after HC administration slightly increased to 1.4fold (2.73 ± 0.76 nmol/lens) at 48 hr, and the levels were much lower than that of "glucose" (44.34 ±0.13 nmol/lens). The effects of PS, CS, and CX on cataract formation and glucose level in lens were determined (Table 3). At the same time, glutathione level in lens was also determined. PS (0.25 /^mol/egg), as well as HC, produced cataract formation, the elevation of glucose, and the decline of glutathione in the lens. However, when 0.25 /Limol/egg of CS or CX was administered, little effect on cataract formation, the elevation of glucose, and the decrease of glutathione could be observed. One Table 3. Relationship between steroid structure and cataractogenesis Lens Dosage Frequency of cataract nmole per egg o II 0 III 0 IV-V 0 0.025 0.25 30 2 6 0 0 2 0.025 0.25 30 2 0 0 CS 0.25 1.0 36 18 CX 0.25 1.0 36 36 HC 2U 48 Ti me ( hr ) 72 100 Fig. 2. Alteration of glucose level in the lens after HC was administered to 15-day-old chick embryos (0 hr). Lenses were removed at the indicated times after HC administration. Glucose content was determined as described in Materials and Methods and calculated as nmoles per lens. Data were expressed as mean ± SE (n = 4-30). In the cases of circles without bars, the bar ranges were smaller than the diameters. Classification of the lenses from HC-treated chick embryos; 100% at stage I at 4 and 10 hr; 30% at stage I and 70% at stage IIIII at 24 hr; 5% at stage I, 5% at stage II—III, and 90% at stage IV-V at 48 hr; 20% at stage I, 45% at stage II—III, and 35% at stage IV-V at 72 hr; 75% at stage I, 19% at stage II—III, and 6% at stage IV-V at 100 hr. Glucose GSH I 36 Control PS Blood Glucose % 100 % 100 % 100 0 32 523 2000 70 42 200 4 0 2 34 753 2270 84 39 240 0 0 0 12 0 6 10 270 98 64 93 0 0 0 0 0 0 46 65 87 78 84 117 Steroid at the indicated dosage was dissolved in 0.2 ml of sterilized water and administered to 15-day-old chick embryos. At 48 hr after steroid administration, lenses and blood were removed. Lenses were classified to stage I to stage V, as described previously.' Data were expressed as number of embryos. The determination of glucose and glutathione in pooled lenses and that of glucose in blood were carried out as described in Materials and Methods. Data were expressed as mean % of control from three experiments for lens and as mean % of control from 5-10 embryos. HC = hydrocortisone hemissucinate sodium, PS = prednisolone hemisuccinate sodium, CS = cortisone hemisuccinate sodium, CX = cortexolone hemisuccinate sodium, GSH = glutathione. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933362/ on 05/07/2017 No. 1 per egg of CS showed some effects, but that of CX did not. In the alteration of blood glucose after glucocorticoid administration, HC and PS showed the elevation, of glucose, but CS and CX did not. Since cortisone and cortexolone are known to have weak or negligible glucocorticoid activity in the developing chick embryo as well as in mammals, 519 ' 20 cataract formation and associated phenomena seemed to be closely related to glucocorticoid activity. It is known that hyperglycemia caused by sugar6"8 or by chemicals9 can induce cataracts in animals. The marked elevation of glucose in the lens after glucocorticoid administration suggests that there are similarities between glucocorticoid-induced cataract and sugar cataract. To study this further, the amount of water, sorbitol, fructose, and glycosylation of protein in control and HC-cataractous lens were determined at 48 hr after HC-treatment (Table 4). Wet weight of control lens and HC-cataractous lens were 6.02 ± 0.04 mg and 5.42 ± 0.1 mg, respectively. The water content in HCcataractous lens was also lower than that in control. Sorbitol and fructose in both control and HC-cataractous lens were undetectable. The amounts of 5-hydroxymethyl-furfural released from the glucosyl moiety in ketoamine linkage of protein were negligible in both control and HC-induced cataractous lenses, with no difference between them. Thus, the data showed no correlation between the extent of sorbitol and cataract formation, and that of glycosylation and cataract formation. It was also interesting to know whether the alteration of glucose level in the lens is directly related to lens opacification. Therefore, we determined the effect of ascorbic acid, which is known to prevent HC-induced cataract formation,3 on the elevation of glucose induced Table 4. Effect of HC-treatment on fructose, sorbitol, and protein glycosylation %of Control Cataract control 6.02 ± 0.04 4.99 ±0.04 82.9 5.42 ±0.10 4.40 ± 0.09 81.2 90.0 88.2 97.9 Fructose/20 lenses Sorbitol/20 lenses nil nil nil nil 5-hydroxymethylfurfural/10 lenses nilf nil* Wet weight (mg/lens)* H2O content (mg/lens) H2O/wet weight (%) 171 ELEVATION OF GLUCOSE IN GLUCOCORTICOID CATARACT / Nishigori er ol. HC (0.25 /zmole/cgg) was administered to 15-day-old chick embryos and 48 hr later their lenses were removed and assayed. * Ten lenses from five embryos were pooled. Their wet weight and dry weight were determined and calculated as per lens. Four more experiments were done, and data were expressed as mean ± SE. Fructose, sorbitol, and 5-hydroxymethylfurfural were determined by the methods described in Materials and Methods. Three experiments were done. t Below 0.4 nmole/9.5 mg protein. $ Below 0.4 nmole/9.1 mg protein. Fig. 3. Glucose content in 50 the lenses obtained from control, HC-treated, and HCascorbic acid (VC)-treated, 40 chick embryo. The 15-dayold, chick embryos treated c with HC (0.25 ^mole/egg) were given ascorbic acid (20 o ^moles/egg) at 3, 10, and 20 E hr after HC administration. c At 48 hr after HC treatment, in 20 the lenses were removed and o o classified. Glucose content 5 was determined and calcu10 lated as nmoles per lens. Data were expressed as mean ± SE (n = 4). The data of HC were obtained from stage IV-V HC HC+VC lenses. The data of HC + VC were obtained from stage I lenses. HC: I (0%), IV-V (94%). HC + VC: I (56%), II—III (24%), IV-V (20%). 1 • - 1 - by HC. As shown in Figure 3, the glucose content of stage I lenses from HC-ascorbic acid treated embryos was lower than that of IV-V lenses from HC-treated embryos, but was still 14-fold higher than the control level. Discussion Clinically, glucocorticoids are among the most valuable drugs for treatment of numerous diseases. However, high-dose or long-term therapy with glucocorticoids is well known to cause side effects. Therefore, it is important to clarify the mechanisms for side effects, such as cataract formation, and to find a preventive procedure against side effects without the loss of therapeutic activities. The mechanism of glucocorticoid-induced cataract formation has not been clarified, although there are several possibilities for direct or indirect effects of glucocorticoid on the lens. As a direct effect, glucocorticoid may produce alterations of metabolic activities in the lens,21"23 or it may react with lens protein, such as crystallin.24"26 Recently, Manabe et al24 demonstrated that, when rat lens was incubated with prednisolone, formation of a prednisolone-lenticular protein adduct and cataract occurred. Bucala et al25 also showed, by injecting steroids into the vitreous chamber of the rabbit eye, that cortisol (hydrocortisone), dexamethasone, and prednisolone could produce cataract, but 17a-hydroprogesterone and 4-pregnen-l 1/3,17a,20«,21-tetrol-3one did not. They suggested the formation of a Schiff base through the amine of lens protein and the C-20 carbonyl of corticoids, followed by a Heys rearrangement with the C-21 hydroxyl to produce a stable ketoamine product. However, it is unlikely in our studies that a Schiff base formation at the C-20 carbonyl in Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933362/ on 05/07/2017 172 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / January 1987 hydrocortisone, prednisolone, and other corticoids was involved in cataract formation. The reasons are as follows. Hydrocortisone, prednisolone, cortisone, and cortexolone, which have in common the 17a-OH and the 17.-0 side chain with the C-20 carbonyl and the C21 hydroxyl groups, were examined in their cataractogenic activity by using their 21-hemisuccinate sodium forms. Hydrocortisone and prednisolone, which have a hydroxyl group at the C-11/3, could produce cataract and cause alterations of lens composition. However, cortisone, which, in chick embryos, is poorly converted to hydrocortisone by reduction at the C-l 1 ketone, 1920 could not produce cataract at the same dose as hydrocortisone and prednisolone, but it showed some effect at 1.0 jmiol/egg. Cortexolone, without the C-l 1/3 hydroxyl in corticoid, at 0.25 and 1.0 /^mol/egg, could not produce cataract. Since the C-l 10 hydroxyl group in the structure of corticoid is essential for glucocorticoid activities,1920 these data demonstrate that cataractogenic activity relates to glucocorticoid activities. Glucocorticoid-induced cataractous lenses in developing chick embryos display at least three common phenomena in the alteration of lens components: an elevation of glucose, a decline of glutathione,'*2 and an elevation of lipid peroxide.27 Glucocorticoid alters metabolic activities in several tissues, which can cause changes in humoral components, such as elevation of blood glucose4 and lipid peroxide.28 It is likely that these substances invade and influence the lens, leading to the loss of transparency. In mammals, adrenocortical hormones have profound effects on glucose and protein metabolism, resulting in the tendency toward hyperglycemia.4 Similar processes may be involved in the elevation of blood glucose level after glucocorticoid administration to developing chick embryos. Glucose is the primary source of metabolic energy in the lens, and is derived from blood via the ocular fluids-aqueous and vitreous humor.29"31 In ovo, the levels of glucose in blood and lens at 48 hr after HC administration were calculated as mM values. In controls, the lenticular level was 0.33 ± 0.04 mM (based on wet weight; Table 4) which was about 1/25 of the blood level. In developing chick embryos with cataract, the concentration in lens and blood increased and became 8.73 ±1.15 mM and 15.80 ± 0.73 mM, respectively. Thus, it was found that, after HC administration, the elevation of lenticular glucose level was marked, and did not occur parallel to that of blood glucose level. "Glucose" level in the lens is also dependent on the metabolism of glucose. In fact, after HC treatment, the metabolism of glucose in the lens is altered; hexokinase activity in HC-cataractous lens was about 80% of control (not shown), although glucose-6-phosphate content in HC-induced cataractous lens was about 1.4-fold above control (possibly through the elevation of "glu- Vol. 28 cose" substrate). Therefore, it would seem that the elevation of "glucose" in the lens after HC administration may be mainly due to the elevation of "glucose" in blood and ocular fluids. Concerning the mechanism of sugar-induced cataract formation, the glycosylation of lenticular protein11-32"35 and the "osmotic theory" caused by accumulation of sorbitol 1011 have been considered. Studies on the lens protein11'32-33 from diabetic and nondiabetic animals indicated that increased glycosylation might contribute to the development of sugar cataract. On the contrary, there have been reports which did not reveal significant differences in glycosylation of lysine in lens crystallins between diabetics and non-diabetics in both animals and humans. 34 ' 35 Piatigorsky et al demonstrated that delta-crystallin from 15-day-old chick embryo lens contains lysine (33.5— 33.7 moles/50000 daltons).36 Therefore, it was interesting to know whether glycosylation of protein occured in glucocorticoid-induced cataract in developing embryos. As shown in results, however, it was found that glycosylation of protein was negligible in HC-induced cataractous lens (below 0.4 nmole/9.1 mg protein), as well as control lens (below 0.4 nmole/9.5 mg protein). Alternatively, it has been hypothesized that, when glucose enters the lens to form a sugar alcohol, it brings in water and changes the ionic balance, which causes swelling and vacuole formation, leading to cataract. 101 ' However, several studies have demonstrated that the onset of either diabetic or galactosemic cataract occurred without affecting the accumulation of polyol, and that diabetic cataracts may be prevented by diets high in fat and protein37 or antioxidant, such vitamin E,38 although there was still an elevation of polyol in the lens to levels similar to those found in untreated rats. In the present studies, we supposed that the synthesis and the accumulation of sorbitol in HC-cataractous lenses could occur, since "glucose" level was markedly accumulated. It was found that vacuoles between cortical and nuclear region were formed in HCinduced cataractous lenses (not shown). However, as demonstrated in the results, sorbitol and fructose could not be detected in HC-induced cataractous lenses, nor in control lenses. Furthermore, dehydration was observed in HC-induced cataractous lenses. The present data indicate that glucocorticoid-induced cataract of developing chick embryos could not be understood, at least by either glycosylation of protein or the elevation of sorbitol. The loss of transparency occurred with the elevation of glucose in the lens, and lens opacity disappeared with a return to control glucose level. It was thought possible that the high content of glucose in the lens leads to the loss of transparency. Ross et al38 have indicated that the glucose, fructose, and sorbitol levels Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933362/ on 05/07/2017 No. 1 ELEVATION OF GLUCOSE IN GLUCOCORTICOID CATARACT / Nishigori er ol. were increased to similarly high or significantly higher levels in the noncataractous lenses of the diabetic rats treated with vitamin E, compared with the cataract lenses of the untreated diabetic animals. They described that sugar cataract was not simply caused by a change of osmolarity. We found that the levels of glucose in stage I of noncataractous lenses from HC-ascorbic acidtreated developing chick embryos was approximately 70% of that of cataractous lenses, but was still around 14-fold above that of control lenses. It was not conceivable that the amount of glucose in the lens was a critical factor between transparency and opacity of lens. It was also found that the glucose in the lenses consisting of 30% of stage I and 70% of stage II—III at 24 hr after HC treatment was almost equal to that in the lenses consisting of 5% of stage I, 5% of stage II—III, and 90% of stage IV-V at 48 hr after HC treatment. These results argue that glucocorticoid-induced cataract could not be caused by osmotic change, depending on glucose level. Thus, glucose elevation may not be directly related to glucocorticoid-induced cataract in developing chick embryos. However, a role for glucose in some aspect of the process cannot be ruled out. Our previous papers have suggested that glucocorticoid-induced cataract formation proceeded via a step of oxidation or peroxidation, since the cataract formation can be suppressed by radical scavengers, such as ascorbic acid,3'27 vitamin E (not shown), and N-(2-mercaptopropionyl)glycine.2 However, the mechanism of production of free radicals in ovo caused by glucocorticoid administration remains obscure. Crabbe hypothesizes that autooxidation of glucose producing free radicals may be involved in sugar cataract.39 Although there are controversial opinions against Crabbe's hypothesis, 939 his hypothesis still seems to be interesting at the present time. However, glucocorticoid has multiple activities in living systems. The effects of one insult or change may be subliminal; when several are combined, they can have a synergistic action in initiating or potentiating a cataractogenic effect. To clarify the mechanism of glucocorticoid-induced cataract formation, other processes must also be considered. Key words: cataract, chick embryo, glucocorticoid, glucose, cortexolone Acknowledgment We are indebted to Dr. D. O. Toft for his advice. References 1. Nishigori H, Lee JW, and Iwatsuru M: An animal model for cataract research: cataract formation in developing chick embryo by glucocorticoid. Exp Eye Res 36:617, 1983. 173 2. Nishigori H, Hayashi R, Lee JW, and Iwatsuru M: Effect of MPG on glucocorticoid-induced cataract formation in developing chick embryo. Invest Ophthalmol Vis Sci 25:1051, 1984. 3. Nishigori H, Hayashi R, Lee JW, Maruyama K, and Iwatsuru M: Preventive effect of ascorbic acid against glucocorticoid-induced cataract formation of developing chick embryo. Exp Eye Res 40:445, 1985. 4. Ensinck JW and Williams RH: Disorder causing hypoglycemia. In Textbook of Endocrinology, 5th ed, Williams RH, editor. Philadelphia, London and Toronto. WB Saunders Company, 1974, pp. 627-659. 5. Koehler DE and Moscona AA: Corticosteroid receptors in the neural retina and other tissues of the chick embryo. Arch Biochem Biophys 170:102, 1975. 6. Sterling RE and Day PL: Blood sugar levels and cataract in Alloxan-treated, galactose-fed and xylose-fed weanling rats. Proc Soc Exp Biol 78:431, 1951. 7. Van Heyningen R: Formation of polyols by the lens of the rat with "sugar" cataract. Nature (London) 184:194, 1959. 8. Patterson JW: Course of diabetes and development of cataracts after injecting dehydroascorbic acid and related substances. Am JPhysiol 165:61, 1951. 9. Kador PF and Kinoshita JH: Diabetic and galactosaemic cataracts. In Human Cataract Formation, Ciba Foundation Symposium 106, Nugent J and Whelan J, editors. London, Pitman Press, 1984, pp. 110-131. 10. Kinoshita JH, Merola LO, and Dikmak E: Osmotic changes in experimental galactose cataracts. Exp Eye Res 1:405, 1962. 11. Cerami A, Stevens VJ, and Monnier VM: Role of nonenzymatic glycosylation in the development of the sequelae of diabetes mellitus. Metabolism 28:431, 1979. 12. Bergmeyer HU, Bernt E, Schmidt F, and Stork H. D-glucose, Determination with hexokinase and glucose-6-phosphate dehydrogenase. In Method of Enzymatic Analysis, 2nd English edition translated from the third German edition, Bergmeyer HU, editor. New York, San Francisco, London, Verlag Chemie Weiheim, Academic Press, 1974, pp. 1196-1201. 13. Bergmeyer HU, Gruber BW, and Gutmann I: D-sorbitol. In Method of Enzymatic Analysis, 2nd English edition translated from the third German edition, Bergmeyer HU, editor. New York, San Francisco, London, Verlag Chemie Weiheim, Academic Press, 1974, pp. 1323-1326. 14. Bernt E and Bergmeyer HU: D-fructose. In Method of Enzymatic Analysis, 2nd English edition translated from the third German edition, Bergmeyer HU, editor. New York, San Francisco, London, Verlag Chemie Weiheim, Academic Press, 1974, pp. 12941322. 15. Fluckiger R and Winterhalter KH: In vitro synthesis of hemoglobin A lc. FEBS Lett 71:356, 1976. 16. Lowry OH, Rosebrough NJ, Fair AL, and Randall RJ: Protein measurement with the folin phenol reagent. J Biol Chem 193: 265, 1951. 17. Sedlak J and Lindsay RH: Estimation of total protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman's reagent. Anal Biochem 25:192, 1968. 18. Upjohn Co: Steroids! Chemical Abstract 53:6302, 1959. 19. Moscona AA and Piddington R: Enzyme induction by corticoids in embryonic cells: Steroid structure and inductive effect. Science 158:496, 1967. 20. Cohen A and Kulka RG: Relationship of steroid structure to induction of chymotrypsinogen in embryonic chick pancreas in vitro. Endocrinology 97:475, 1975. 21. Mayman CI, Miller D, and Tijerina MC: In vitro production of steroid cataract in bovine lens. II: Measurement of sodium potassium adenosine triphosphate activity. Acta Ophthalmol 57: 1107, 1979. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933362/ on 05/07/2017 174 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / January 1987 22. Greiner JV, Kopp SJ, and Glonek T: Dynamic changes in the organophosphate profile upon treatment of the crystalline lens with dexamethasone. Invest Ophthalmol Vis Sci 23:14, 1982. 23. 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