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The increase in lipid peroxidation in diabetic rat lens can be reversed by oral sorbinil
‘The Increase
in Lipid Peroxidation in Diabetic by Oral Sorbinil
Rat Lens Can Be Reversed
Li-An Yeh and Michael A. Ashton Malondialdehyde (MDA), a product of lipid peroxidation, was measured in the lens of rats in which diabetes had been induced by treatment with streptozotocin (85 mg/kg). The MDA level increases nearly 100% in lenses from diabetic rats, compared with lenses from age-matched control rats. Treating rats with insulin decreases the MDA content of the diabetic rat lens to normal levels. This suggests that the increase in MDA of diabetic rats lens is not due to a toxic effect of streptozotocin, but instead is the result of diabetes mellitus or insulin deficiency. Administration of the aldose reductase inhibitor, sorbinil, decreases lens MDA levels in diabetic rats in a dose-dependent manner with a median effective dose (ED,) of 7.5 mg/kg. This study demonstrates that sorbinil can prevent lipid peroxidation of rat lens resulting from insulin deficiency, which can aid in helping the lens to cope with the oxidative stress of diabetes. 0 1990 by W.B. Saunders Company.
T
HE MOLECULAR MECHANISM of senile cataractogenesis has been proposed to be oxidative stress.‘-3 Hydrogen peroxide, which is one of the toxic metabolites of oxygen, increases significantly (approximately twofold to threefold) in several experimental cataract models4-6and also in human senile cataracts.7,8 It has been suggested that peroxidation of lenticular plasma membrane lipids could be one of the initiating events in the pathogenesis of cataract formation.9~” Malondialdehyde (MDA), a major product of lipid peroxidation, is significantly increased by twofold to fourfold in human senile cataracts.‘.’ The increases of MDA content in the cataractous lenses are accompanied by a drastic increase in the hydrogen peroxide level in the aqueous humor of the eye in both human and experimental cataracts.‘.” The correlation between the levels of MDA in the lens and levels of H,O, in aqueous and vitreous humor has been reviewed extensively elsewhere.“’ Glutathione (GSH) is thought to be the most important antioxidant in the lens. A decrease in lens GSH has been found in most experimental cataract models and also in human cataracts.‘2-‘7 It has been reported by several investigators that lenticular GSH is dramatically decreased in streptozotocin-induced diabetic rats.‘2”“ This decrease in GSH levels, which likely renders the lens more susceptible to oxidative stress, could be one of the causes for the early onset of senile cataract in diabetic humans.“-” The aldose reductase inhibitor sorbinil, has been reported to normalize the decreased glutathione level in the lens of diabetic rat.12’14The mechanism by which sorbinil restored lenticular glutathione level is unclear. However, it has been speculated that the blockage of the polyol pathway leads to normal glycolysis and hexose monophosphate shunt activity, which then restores adenosine triphosphate (ATP) levels to normal in the lens, since total glutathione biosynthesis is ATP-dependent. The purpose of this study was to test if the aldose reductase inhibitor, sorbinil, could decrease the oxidative stress rendered by diabetes. Levels of MDA, a product derived from the breakdown of the lipid peroxides, were used as an index of peroxidative degradation of the lipid, and thus a measure of oxidative stress. MATERIALS AND METHODS
Animal Studies Male Sprague-Dawley rats weighing approximately 200 g (Charles River Laboratories, Wilmington, MA) were maintained with free
Mefabolism, Vol39,
No 6 (June). 1990: pp 6 19-622
access to food (Prolab R-M-H 3000, Agway, Syracuse, NY) and water. Diabetes was induced by injection of streptozotocin (85 mg/kg body weight, Upjohn Chemical, Kalamazoo, MI) in 0.01 mol/L citrate buffer, pH 4.5, into the tail vein of fed rats. This dose of streptozotocin produced severe hyperglycemia, with blood glucose levels ranging between 400 and 550 mg/dL. Sorbinil (CP-45,634, Pfizer, Groton, CT) was administered as an aqueous suspension by oral gavage at the indicated times and doses. The animals were killed and the eyes were removed immediately and the lenses were dissected free from the eyeglobe using a posterior approach.
Tissue Preparation and MDA Determination MDA was assayed according to Ohkawa et al’” with slight modifications. Lenses were homogenized in a volume of one-tenth lens weight of 6% perchloric acid at O°C with a glass homogenizer and then centrifuged in an Eppendorf microfuge for 15 minutes at 4“C. Two hundred microliters of the supernatant was added to 42 PL of 5-mol/L sodium acetate to obtain a final pH of 3.5. Two hundred microliters each of 8.1% sodium dodecyl sulfate (SDS) and 0.8% thiobarbituric acid (TBA) were then added to the above mixture, and water was added to a final volume of 1 mL. The samples are then placed into an heating block at 95OC for 50 minutes, and then cooled to room temperature with tap water. Distilled water, 0.5 mL, and 2.5 mL mixture of butanol and pyridine (15:l) were then added to each sample, vortexed and centrifuged at 3,000 rpm for 15 minutes. Two milliliters of the top layer were removed and read fluorimetrically at 5 15 nm excitation and 553 nm emission. 1,1,3,3,Tetraethoxypropane (TEP) was used as a standard. The tissue preparation yielded a 75% to 80% MDA recovery, and sorbinil had no interference with the assay. RESULTS
Eflects of Diabetes on Lenticular
MDA
Level
Previous studies have indicated that MDA levels are elevated in experimental cataract models and in human cataracts.” The rat lens MDA levels at 24 hours, 2 weeks, and 18 weeks after streptozotocin (85 mg/kg intravenously [IV]) administration were compared with age-matched controls. There was an immediate 35% increase in MDA From the Department of Metabolic Diseases, Central Research Division, Pfizer Inc. Groton, CT. Address reprint requests to Li-An Yeh, PhD, Department of Metabolic Diseases, Central Research Division, Pfizer Inc. Groton. CT 06340. o 1990 by W.B. Saunders Company. 0026-0495/90/3906-0013%03.00/0 619
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620
formation in diabetic lens compared with the controls at 24 hours after streptozotocin was injected. By 2 weeks, the MDA increased by nearly 100% in diabetic lens compared with normal rat lens. The increase in MDA levels persisted to 18 week of diabetic state as shown in Fig 1. These data indicate that lipid peroxidation may be one of the initial events that triggers the cataract formation.
600 r
ST2
Insulin
Effects of Insulin on Lenticular MDA Level Several studies have suggested that the mechanism of streptozotocin-induced damage in the rat B cell is mediated through oxygen free radical formation.‘9-24 It is thus possible that the increase in lipid peroxidation is the consequence of streptozotocin-induced damage, rather than the result of the pathological state of diabetes. In order to test this possibility, insulin was administered to rats at 3 days post-streptozotocin treatment and blood glucose of the rats was monitored. As shown in Fig 2, the blood glucose of diabetic rats was elevated to 550 mg/dL 1 day after streptozotocin (85 mg/kg IV) injection, and reached 700 mg/kg at day 13. However, the blood glucose in diabetic rats that had been treated with insulin (10 U/rat) daily beginning at day 3, decreased to 150 mg/dL by day 7 and remained at normal levels throughout the experimental period. At day 14, all the rats were killed, and the MDA levels in the lens were determined as shown in Fig 3. The MDA content in the lens of diabetic rats treated with insulin was not significantly different from the MDA level of the lens from normal control rats. This experiment indicates the increase in lens MDA in the diabetic rat is due to metabolic milieu and, in particular, insulin deficiency or glucose elevation in the rat rather than a toxic effect of streptozotocin. Effects of Sorbinil on Lenticular MDA Level
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Fig 2. The effect of insulin on blood glucose levels of diabetic rats. Rats were made diabetic as described in the Material and Methods section. Three days after diabetic state was induced, insulin was administered IV daily to one group of rats at 10 U/d. A-A, Blood glucose levels were monitored every other day starting 24 hours after diabetes induction. o--O, untreated diabetic rats were run in parallel experiments; O--O, agematched control rats are also included. Each value is a mean of 18 to 20 determinations (n = 18-20).
in MDA formation in diabetic rat lens with a median effective dose (ED,,) of 7.5 mg/kg. The ED,, of sorbinil to reverse the increase MDA level is in the close range of the ED,, (2.5 mg/kg) of sorbinil found to reverse the decrease in glutathione levels in the diabetic rat lens.14 DISCUSSION
Previous studies have indicated that several metabolic changes due to the diabetic state of rats can be reversed by the aldose reductase inhibitor, sorbinil, in a dose-dependent To determine if sorbinil has any effect on MDA manner. 12-14 levels of diabetic rat lens, diabetes was induced in rats for 1 week and the rats were then gavaged orally with sorbinil once a day for 7 days, at the doses of 2.5, 10, and 25 mg/kg. As shown in Fig 4, sorbinil produced a dose-dependent decrease
120'
IOOl
ADMINISTRATION
Fig 1. Lens MDA content in diabetic rats. Rats ware made diabetic by injection of streptozotocin (86 mg/kgl end lens MDA level wes determined as described in the Material and Methods. Values of MDA content are expressed as percentage of MDA content in the non-diabetic control rat lens.
Most types of cataracts are characterized by a dramatic decrease in the content of GSH levels in the lens.25-30 Therefore, it appears that GSH may play an important role in maintaining lens transparency. GSH may act either directly as a protective agent by scavenging oxygen derived free radicals or by protecting other reactive thiols of crystallines, enzymes, or membrane proteins and lipids. It is believed that peroxidation of lenticular plasma membrane lipids may be one of the initiating events in the pathogenesis of cataract. However, details concerning the oxidants and reactions that trigger in the initiation of cataract formation are not well understood. The present study shows that in 2-week diabetic rat lens there is a significant increase in MDA formation, an end product of lipid peroxidation. The increase in MDA in diabetic lens can be prevented by treating the rats with an antioxidant, BHT (butylated hydroxytoluene at 300 mg/kg) (Morehouse and Ashton, manuscript in preparation). It was also reported by Linklater et al that oral BHT treatment could reduce protein leakage from lenses in diabetic rats.3’ These data further support the fact that MDA elevation is due to lipid peroxidation. This increase in lipid peroxidation can be reversed in a doseresponsive fashion by treating the diabetic animal with insulin or an aldose reductase inhibitor, sorbinil. Since insulin treatment can reverse the elevation in MDA levels
SORBINOL REVERSED OXIDATIVE
STRESS IN DIABETIC LENS
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GROUP Fig 3. Effect of insulin on lens MDA level of diabetic rats. The experimental conditions are the same as described in Fig 2. At the end of experiment, rats were killed and the MDA levels in the lens were determined as described in Materiel and Methods. Group 1, age-matched control rats; group 2, untreated diabetic rats; group 3, diabetic rats treated with insulin. Each value is a mean of 18 to 20 determinations. The value for diabetic animels is significantly different from normal control value at confidence level of lP < .OOl. The value for diabetic animals treated with insulin is not significantly different from normal control values at confidence levels of tP < .4. The value for diebetic animals treated with insulin (group 3) is significantly diierent from untreated diabetic animals (group 2) at confidence level of $P < ~301.
subsequent to initiation of diabetes in rats, lipid peroxidation is unlikely to be a consequence of a streptozotocin-induced toxicity. Since sorbinil has no effect on either blood glucose level32or acts as an antioxidant,33 but has an effect similar to insulin on lenticalur MDA levels, there may be a link between the polyol pathway and lipid peroxidation. Other aldose reductase inhibitors that are structurely different from sorbinil (carboxylic acids series v hydantoins) tested in our rat model, have shown similar inhibitory activity as sorbinil in preventing MDA increase in diabetic rat lens (Yeh and Ashton, unpublished results). Our dose-response study indicated an increase in MDA formation and H,O, concentrations in the medium is parallel with the cataract formation. Sorbinil addition could partially prevent both activities (Yeh and Ashton, manuscript in preparation). The exact mechanism by which sorbinil can normalize the MDA levels in diabetic rat lens is unknown. However, it has been demonstrated by us and other investigators’2-‘4 that there is a dramatic decrease in lens glutathione and ATP levels in 2-week diabetic rats, and that sorbinil treatment can reverse those deficiencies in diabetic lens. GSH is thought to be the most important antioxidant in the lens. The increase in lipid peroxidation reflected by elevated MDA level in the experimental diabetic rat lens may be secondary to the decrease in
Fig 4. The effect of sorbinil on the MDA content of lens from the 2-week diabetic rat. Rats were made diabetic as described in Material and Methods. One week later rats received sorbinil by oral gavage at indicated doses. One week after the sorbinil treatment, rats were killed and lens MDA levels were determined as described in Material and Methods. Group 1. age-matched normal control; group 2, untreated diabetic control; group 3, 2.6 mg/kg sorbinil; group 4, 10 mg/kg sorbinol, group 6, 26 mg/kg sorbinol. Data are expressed as the mean of four separate determinations. All values for diabetic animals are significantly different from normal control value at confidence level indicated, lP <.OOl, tP ~05; except where noted, all values for sorbiniltreated animals are significantly different from untreeted diabetic values at confidence levels indicated; #P < .OB, §P < .02, /P < .OOl
lens glutathione and ATP content. The ED,, of sorbinil for decreasing elevated lens MDA is close to the ED,, for restoration of GSH levels (7.5 mg/kg v 2.5 mg/kg),14 suggesting that there is a link between an elevation of MDA and a depression of lenticular GSH and ATP. A recent study by Lou et a134investigated the causes for drastic depletion of GSH in the lens of galactosemic and diabetic rats, and suggested that total GSH biosynthesis is impaired by leaky membrane as initiated by polyol pathway. The aldose reductase inhibitor, sorbinil, blocks the polyol pathway, thereby preventing the loss of ATP and amino acids due to activation of hexomonophosphate shunt activity and leaky membrane, leading to the restoration of GSH levels and reducing equivalents in the lens to overcome oxidative stress. Other investigators have also suggested a similar antioxidant role for sorbinil.‘2,‘3 Ohkawa et al’* reported that plasma levels of MDA in diabetic patients are significantly higher than those of normal subjects, and are higher in patients with angiopathy than in patients without angiopathy. Thus, sorbinil might protect against diabetic cataract in humans as a result of reducing damage due to oxidative stress. ACKNOWLEDGMENT
The authors wish to thank Drs B.L. Mylari, Hutson, and C. Harbert for their comments.
R. Sarges,
N.J.
REFERENCES
1. Spector A: The search for a solution to senile cataract. Invest Ophthalmol Vis Sci 25:130-146, 1984 2. Chylack LT Jr: Mechanisms of senile cataract formation. Ophthalmology 9 1596-602, 1984
3. Bhuyan KC, Bhuyan DK: Molecular mechanism of cataractogenesis. Curr Eye Res 3:67-8 1, 1984 4. Bhuyan KC, Bhuyan DK, Turtz AI: Aminotriazole effect on the ocular tissues in rabbit I.R.C.S. Med Sci 2:1594, 1974
622
5. Bhuyan KC, Bhuyan DK: Regulation of hydrogen peroxide in eye humor, effect of 3-amino-1-H-1,2,4-triazole on catalase and glutathione peroxidase of rabbit eye. Biochim Biophys Acta 497:641651.1977 6. Bhuyan KC, Bhuyan DK: Superoxide dismutase of the eye: Relative functions of superoxide dismutase and catalase in protecting the ocular lens from oxidative damage. Biochim Biophys Acta 542:28-38,1978 7. Spector A, Garner WH: Hydrogen peroxide and human cataract. Exp Eye Res 33:673, 198 1 8. Tomba MC, Gandolfi SA, Maraini G: Search for an oxidative stress in human senile cataract. Hydrogen peroxide and ascorbic acid in the aqueous humour and malondialdehyde in the lens. Lens Res 21263-276, 1985 9. Bhuyan KC, Master RWP, Coles R, et al: Adduct in human senile cataract. Mech Ageing Dev 34:289-296, 1986 10. Bhuyan KC, Bhuyan DK: Molecular mechanism of cataractogenesis (3): Toxic metabolites of oxygen as initiators of lipid peroxidation and cataract. Curr Eye Res 3:67-81, 1984 11. Bhuyan KC, Bhuyan DC, Kuck JFR Jr, et al: Increased lipid peroxidation and altered membrane functions in emory mouse cataract. Curr Eye Res 2597-606, 1983 12. Gonzalez AM, Sochor M, McLean P: The effect of an aldose reductase inhibitor (sorbinil) on the level of metabolites in lenses of diabetic rats. Diabetes 32:482-485, 1983 13. Gonzalez AM, Sochor M, Hothersall JS, et al: Effect of aldose reductase inhibitor (sorbinil) on integration of polyol pathway, pentose phosphate pathway and glycolytic route in diabetic rat lens. Diabetes 35:1200-1205, 1986 14. Yeh LA, Rafford C, Guddu KJ, et al: Na,K-ATPase pumping activity is not directly linked to myo-inositol levels after sorbinil treatment in lenses of diabetic rats. Diabetes 36:1414-1419, 1987 15. Clayton RM, Cuthbert J, Seth J, et al: Human cataract formation: Epidemiological and other studies in the assessment of factors contributing to cataractogenesis, Pitman, London, Ciba Foundation Symposium 106:25-47, 1984 16. Varma SD: Aldose reductase and the etiology of diabetic cataracts. Curr Top Eye Res 3:91-155, 1980 17. Ederer F, Hiller R, Taylor H: Senile lens changes and diabetes in two population studies. Am J Ophthalmol 91:381-395, 1981 18. Ohkawa H, Ohishi N, Yagi K: Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal B&hem 95:351358,1979 19. Heikkila RE, Cabbat FS: Protection against alloxan-induced diabetes in mice by the hydroxyl radical scavenger dimethylurea. Eur J Pharmacol52:57-60, 1978
YEH AND ASHTON
20. Tibaldi J, Benjamin J, Cababat FS, et al: Protection against alloxan-induced diabetes by various urea derivatives: Relationship between protective effects and reactivity with the hydroxyl radical. J Pharmacol ExpTher 211:415-418,1979 21. Grankvist K, Marklund S, Sehlin J, et al: Superoxide dismutase, catalase and scavengers of hydroxyl radicals protect against the toxic action of alloxan on pancreatic islet cells in vitro. Biochem J 182:17-25, 1979 22. Fischer LJ, Hamburger SA: Inhibition of alloxan action in isolated pancreatic islets by superoxide dismutase, catalase and a metal chelator. Diabetets 29:213-216, 1980 23. Fischer LJ, Hamburger SA: Dimethylurea: A radical scavenger that protects isolated pancreatic islets from the effects of alloxan and dihydroxy fumarate exposure. Life Sci 26:1405-1409, 1980 24. Wilson GL, Patton NJ, McCord JM, et al: Mechanisms of streptozotocin- and alloxan-induced damage in rat B cells. Diabetologia 27:587-591, 1984 25. Frieburg D, Manthey KF: Glutathione and NADP linked enzymes in human senile cataract. Exp Eye Res 15:173-187, 1973 26. Anderson E, Spector A: The state of sulfhydryl groups in normal and cataractous human lens proteins in nuclear region. Exp Eye Res 26:407-417, 1978 27. Kuck JFR, Kuck KD: The emory mouse cataract: Loss of soluble protein, glutathione, protein sulfhydryl and other changes. Exp Eye Res 36:351-362, 1983 28. Megaw JM: Gluthathione and ocular photobiology. Curr Eye Res 3:83-87, 1984 29. Calvin HI, Medvedovsky C, Worgul BV: Near-total glutathion depletion and age-specific cataracts induced by buthionine sulfoximine in mice. Science 233:553-555, 1986 30. Taura T, Giblin FJ, Reddy VN: Effects of glutathione depletion on lens membrane permeability: A histochemical study usine ruthernium red. Lens Res 3:93-106, 1986 31. Linklater HA, Dzialoszynski T, Mcleod HL, et al: Modelling cortical cataractogenesis VIII: Effects of butylated hydroxytoluene (BHT) in reducing protein leakage from lenses in diabetic rats. Exp Eye Res 43:305-313, 1986 32. Peterson MJ, Sarges R, Aldinger CE, et al: CP-45,634: A novel aldose reductase inhibitor that inhibits polyol pathway activity in diabetic and galactosemic rats. Metabolism 28:456-461, 1979 (SUPPI) 33. Kador PF, Kinoshita JH, Brittain DR, et al: Purified rat lens aldose reductase. Biochem J 240:233-237, 1986 34. Lou MF, Dickerson JE Jr, Garadi R, et al: Glutathione depletion in the lens of galactosemic and diabetic rats. Exp Eye Res 46517-530, 1988
in Lipid Peroxidation in Diabetic by Oral Sorbinil
Rat Lens Can Be Reversed
Li-An Yeh and Michael A. Ashton Malondialdehyde (MDA), a product of lipid peroxidation, was measured in the lens of rats in which diabetes had been induced by treatment with streptozotocin (85 mg/kg). The MDA level increases nearly 100% in lenses from diabetic rats, compared with lenses from age-matched control rats. Treating rats with insulin decreases the MDA content of the diabetic rat lens to normal levels. This suggests that the increase in MDA of diabetic rats lens is not due to a toxic effect of streptozotocin, but instead is the result of diabetes mellitus or insulin deficiency. Administration of the aldose reductase inhibitor, sorbinil, decreases lens MDA levels in diabetic rats in a dose-dependent manner with a median effective dose (ED,) of 7.5 mg/kg. This study demonstrates that sorbinil can prevent lipid peroxidation of rat lens resulting from insulin deficiency, which can aid in helping the lens to cope with the oxidative stress of diabetes. 0 1990 by W.B. Saunders Company.
T
HE MOLECULAR MECHANISM of senile cataractogenesis has been proposed to be oxidative stress.‘-3 Hydrogen peroxide, which is one of the toxic metabolites of oxygen, increases significantly (approximately twofold to threefold) in several experimental cataract models4-6and also in human senile cataracts.7,8 It has been suggested that peroxidation of lenticular plasma membrane lipids could be one of the initiating events in the pathogenesis of cataract formation.9~” Malondialdehyde (MDA), a major product of lipid peroxidation, is significantly increased by twofold to fourfold in human senile cataracts.‘.’ The increases of MDA content in the cataractous lenses are accompanied by a drastic increase in the hydrogen peroxide level in the aqueous humor of the eye in both human and experimental cataracts.‘.” The correlation between the levels of MDA in the lens and levels of H,O, in aqueous and vitreous humor has been reviewed extensively elsewhere.“’ Glutathione (GSH) is thought to be the most important antioxidant in the lens. A decrease in lens GSH has been found in most experimental cataract models and also in human cataracts.‘2-‘7 It has been reported by several investigators that lenticular GSH is dramatically decreased in streptozotocin-induced diabetic rats.‘2”“ This decrease in GSH levels, which likely renders the lens more susceptible to oxidative stress, could be one of the causes for the early onset of senile cataract in diabetic humans.“-” The aldose reductase inhibitor sorbinil, has been reported to normalize the decreased glutathione level in the lens of diabetic rat.12’14The mechanism by which sorbinil restored lenticular glutathione level is unclear. However, it has been speculated that the blockage of the polyol pathway leads to normal glycolysis and hexose monophosphate shunt activity, which then restores adenosine triphosphate (ATP) levels to normal in the lens, since total glutathione biosynthesis is ATP-dependent. The purpose of this study was to test if the aldose reductase inhibitor, sorbinil, could decrease the oxidative stress rendered by diabetes. Levels of MDA, a product derived from the breakdown of the lipid peroxides, were used as an index of peroxidative degradation of the lipid, and thus a measure of oxidative stress. MATERIALS AND METHODS
Animal Studies Male Sprague-Dawley rats weighing approximately 200 g (Charles River Laboratories, Wilmington, MA) were maintained with free
Mefabolism, Vol39,
No 6 (June). 1990: pp 6 19-622
access to food (Prolab R-M-H 3000, Agway, Syracuse, NY) and water. Diabetes was induced by injection of streptozotocin (85 mg/kg body weight, Upjohn Chemical, Kalamazoo, MI) in 0.01 mol/L citrate buffer, pH 4.5, into the tail vein of fed rats. This dose of streptozotocin produced severe hyperglycemia, with blood glucose levels ranging between 400 and 550 mg/dL. Sorbinil (CP-45,634, Pfizer, Groton, CT) was administered as an aqueous suspension by oral gavage at the indicated times and doses. The animals were killed and the eyes were removed immediately and the lenses were dissected free from the eyeglobe using a posterior approach.
Tissue Preparation and MDA Determination MDA was assayed according to Ohkawa et al’” with slight modifications. Lenses were homogenized in a volume of one-tenth lens weight of 6% perchloric acid at O°C with a glass homogenizer and then centrifuged in an Eppendorf microfuge for 15 minutes at 4“C. Two hundred microliters of the supernatant was added to 42 PL of 5-mol/L sodium acetate to obtain a final pH of 3.5. Two hundred microliters each of 8.1% sodium dodecyl sulfate (SDS) and 0.8% thiobarbituric acid (TBA) were then added to the above mixture, and water was added to a final volume of 1 mL. The samples are then placed into an heating block at 95OC for 50 minutes, and then cooled to room temperature with tap water. Distilled water, 0.5 mL, and 2.5 mL mixture of butanol and pyridine (15:l) were then added to each sample, vortexed and centrifuged at 3,000 rpm for 15 minutes. Two milliliters of the top layer were removed and read fluorimetrically at 5 15 nm excitation and 553 nm emission. 1,1,3,3,Tetraethoxypropane (TEP) was used as a standard. The tissue preparation yielded a 75% to 80% MDA recovery, and sorbinil had no interference with the assay. RESULTS
Eflects of Diabetes on Lenticular
MDA
Level
Previous studies have indicated that MDA levels are elevated in experimental cataract models and in human cataracts.” The rat lens MDA levels at 24 hours, 2 weeks, and 18 weeks after streptozotocin (85 mg/kg intravenously [IV]) administration were compared with age-matched controls. There was an immediate 35% increase in MDA From the Department of Metabolic Diseases, Central Research Division, Pfizer Inc. Groton, CT. Address reprint requests to Li-An Yeh, PhD, Department of Metabolic Diseases, Central Research Division, Pfizer Inc. Groton. CT 06340. o 1990 by W.B. Saunders Company. 0026-0495/90/3906-0013%03.00/0 619
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620
formation in diabetic lens compared with the controls at 24 hours after streptozotocin was injected. By 2 weeks, the MDA increased by nearly 100% in diabetic lens compared with normal rat lens. The increase in MDA levels persisted to 18 week of diabetic state as shown in Fig 1. These data indicate that lipid peroxidation may be one of the initial events that triggers the cataract formation.
600 r
ST2
Insulin
Effects of Insulin on Lenticular MDA Level Several studies have suggested that the mechanism of streptozotocin-induced damage in the rat B cell is mediated through oxygen free radical formation.‘9-24 It is thus possible that the increase in lipid peroxidation is the consequence of streptozotocin-induced damage, rather than the result of the pathological state of diabetes. In order to test this possibility, insulin was administered to rats at 3 days post-streptozotocin treatment and blood glucose of the rats was monitored. As shown in Fig 2, the blood glucose of diabetic rats was elevated to 550 mg/dL 1 day after streptozotocin (85 mg/kg IV) injection, and reached 700 mg/kg at day 13. However, the blood glucose in diabetic rats that had been treated with insulin (10 U/rat) daily beginning at day 3, decreased to 150 mg/dL by day 7 and remained at normal levels throughout the experimental period. At day 14, all the rats were killed, and the MDA levels in the lens were determined as shown in Fig 3. The MDA content in the lens of diabetic rats treated with insulin was not significantly different from the MDA level of the lens from normal control rats. This experiment indicates the increase in lens MDA in the diabetic rat is due to metabolic milieu and, in particular, insulin deficiency or glucose elevation in the rat rather than a toxic effect of streptozotocin. Effects of Sorbinil on Lenticular MDA Level
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Fig 2. The effect of insulin on blood glucose levels of diabetic rats. Rats were made diabetic as described in the Material and Methods section. Three days after diabetic state was induced, insulin was administered IV daily to one group of rats at 10 U/d. A-A, Blood glucose levels were monitored every other day starting 24 hours after diabetes induction. o--O, untreated diabetic rats were run in parallel experiments; O--O, agematched control rats are also included. Each value is a mean of 18 to 20 determinations (n = 18-20).
in MDA formation in diabetic rat lens with a median effective dose (ED,,) of 7.5 mg/kg. The ED,, of sorbinil to reverse the increase MDA level is in the close range of the ED,, (2.5 mg/kg) of sorbinil found to reverse the decrease in glutathione levels in the diabetic rat lens.14 DISCUSSION
Previous studies have indicated that several metabolic changes due to the diabetic state of rats can be reversed by the aldose reductase inhibitor, sorbinil, in a dose-dependent To determine if sorbinil has any effect on MDA manner. 12-14 levels of diabetic rat lens, diabetes was induced in rats for 1 week and the rats were then gavaged orally with sorbinil once a day for 7 days, at the doses of 2.5, 10, and 25 mg/kg. As shown in Fig 4, sorbinil produced a dose-dependent decrease
120'
IOOl
ADMINISTRATION
Fig 1. Lens MDA content in diabetic rats. Rats ware made diabetic by injection of streptozotocin (86 mg/kgl end lens MDA level wes determined as described in the Material and Methods. Values of MDA content are expressed as percentage of MDA content in the non-diabetic control rat lens.
Most types of cataracts are characterized by a dramatic decrease in the content of GSH levels in the lens.25-30 Therefore, it appears that GSH may play an important role in maintaining lens transparency. GSH may act either directly as a protective agent by scavenging oxygen derived free radicals or by protecting other reactive thiols of crystallines, enzymes, or membrane proteins and lipids. It is believed that peroxidation of lenticular plasma membrane lipids may be one of the initiating events in the pathogenesis of cataract. However, details concerning the oxidants and reactions that trigger in the initiation of cataract formation are not well understood. The present study shows that in 2-week diabetic rat lens there is a significant increase in MDA formation, an end product of lipid peroxidation. The increase in MDA in diabetic lens can be prevented by treating the rats with an antioxidant, BHT (butylated hydroxytoluene at 300 mg/kg) (Morehouse and Ashton, manuscript in preparation). It was also reported by Linklater et al that oral BHT treatment could reduce protein leakage from lenses in diabetic rats.3’ These data further support the fact that MDA elevation is due to lipid peroxidation. This increase in lipid peroxidation can be reversed in a doseresponsive fashion by treating the diabetic animal with insulin or an aldose reductase inhibitor, sorbinil. Since insulin treatment can reverse the elevation in MDA levels
SORBINOL REVERSED OXIDATIVE
STRESS IN DIABETIC LENS
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GROUP
3
GROUP Fig 3. Effect of insulin on lens MDA level of diabetic rats. The experimental conditions are the same as described in Fig 2. At the end of experiment, rats were killed and the MDA levels in the lens were determined as described in Materiel and Methods. Group 1, age-matched control rats; group 2, untreated diabetic rats; group 3, diabetic rats treated with insulin. Each value is a mean of 18 to 20 determinations. The value for diabetic animels is significantly different from normal control value at confidence level of lP < .OOl. The value for diabetic animals treated with insulin is not significantly different from normal control values at confidence levels of tP < .4. The value for diebetic animals treated with insulin (group 3) is significantly diierent from untreated diabetic animals (group 2) at confidence level of $P < ~301.
subsequent to initiation of diabetes in rats, lipid peroxidation is unlikely to be a consequence of a streptozotocin-induced toxicity. Since sorbinil has no effect on either blood glucose level32or acts as an antioxidant,33 but has an effect similar to insulin on lenticalur MDA levels, there may be a link between the polyol pathway and lipid peroxidation. Other aldose reductase inhibitors that are structurely different from sorbinil (carboxylic acids series v hydantoins) tested in our rat model, have shown similar inhibitory activity as sorbinil in preventing MDA increase in diabetic rat lens (Yeh and Ashton, unpublished results). Our dose-response study indicated an increase in MDA formation and H,O, concentrations in the medium is parallel with the cataract formation. Sorbinil addition could partially prevent both activities (Yeh and Ashton, manuscript in preparation). The exact mechanism by which sorbinil can normalize the MDA levels in diabetic rat lens is unknown. However, it has been demonstrated by us and other investigators’2-‘4 that there is a dramatic decrease in lens glutathione and ATP levels in 2-week diabetic rats, and that sorbinil treatment can reverse those deficiencies in diabetic lens. GSH is thought to be the most important antioxidant in the lens. The increase in lipid peroxidation reflected by elevated MDA level in the experimental diabetic rat lens may be secondary to the decrease in
Fig 4. The effect of sorbinil on the MDA content of lens from the 2-week diabetic rat. Rats were made diabetic as described in Material and Methods. One week later rats received sorbinil by oral gavage at indicated doses. One week after the sorbinil treatment, rats were killed and lens MDA levels were determined as described in Material and Methods. Group 1. age-matched normal control; group 2, untreated diabetic control; group 3, 2.6 mg/kg sorbinil; group 4, 10 mg/kg sorbinol, group 6, 26 mg/kg sorbinol. Data are expressed as the mean of four separate determinations. All values for diabetic animals are significantly different from normal control value at confidence level indicated, lP <.OOl, tP ~05; except where noted, all values for sorbiniltreated animals are significantly different from untreeted diabetic values at confidence levels indicated; #P < .OB, §P < .02, /P < .OOl
lens glutathione and ATP content. The ED,, of sorbinil for decreasing elevated lens MDA is close to the ED,, for restoration of GSH levels (7.5 mg/kg v 2.5 mg/kg),14 suggesting that there is a link between an elevation of MDA and a depression of lenticular GSH and ATP. A recent study by Lou et a134investigated the causes for drastic depletion of GSH in the lens of galactosemic and diabetic rats, and suggested that total GSH biosynthesis is impaired by leaky membrane as initiated by polyol pathway. The aldose reductase inhibitor, sorbinil, blocks the polyol pathway, thereby preventing the loss of ATP and amino acids due to activation of hexomonophosphate shunt activity and leaky membrane, leading to the restoration of GSH levels and reducing equivalents in the lens to overcome oxidative stress. Other investigators have also suggested a similar antioxidant role for sorbinil.‘2,‘3 Ohkawa et al’* reported that plasma levels of MDA in diabetic patients are significantly higher than those of normal subjects, and are higher in patients with angiopathy than in patients without angiopathy. Thus, sorbinil might protect against diabetic cataract in humans as a result of reducing damage due to oxidative stress. ACKNOWLEDGMENT
The authors wish to thank Drs B.L. Mylari, Hutson, and C. Harbert for their comments.
R. Sarges,
N.J.
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