* Medical Sciences Program, Indiana University School of Medicine, Bloomington, Indiana 47405-7005; and
Alcon Laboratories, Fort Worth, Texas
Received April 1, 1999; accepted June 29, 1999
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ABSTRACT |
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Key Words: catalase; glutathione reductase; superoxide dismutase; lipid peroxidation; reactive oxygen species; aldose reductase inhibitors.
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INTRODUCTION |
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Aldose reductase, an enzyme whose physiological role is not totally understood, converts glucose to sorbitol in the first and rate-limiting step of the polyol pathway (Kinoshita et al., 1990; Raskin and Rosenstock, 1987
). Competitive inhibition of aldose reductase (Petrash et al., 1994
) impedes formation of diabetic cataracts, due to sorbitol flux within the lens fibers (Giugliano and Ceriello, 1996
; Tomlinson et al., 1994
). Thus, in the crystalline lens as in renal medulla (Wu et al., 1993
), aldose reductase has an osmoregulatory function. In vascular tissues such as kidney glomerulus, nerve, and retina, where sorbitol accumulation alone cannot account for the observed pathology (Boel et al., 1995
), aldose reductase may have another physiological role. For instance, aldose reductase mRNA is induced by several different oxidative stressors, suggesting that aldose reductase itself may be involved in cellular antioxidant defense mechanisms (Spycher et al., 1997
).
Aldose reductase inhibitors distribute to liver and other organs after topical ocular administration, causing inhibition of lenticular aldose reductase and renal L-hexonate dehydrogenase (Sastry et al., 1995). Aldose reductase inhibitors AL-1576 and AL-4114 induce some hepatic biotransformation enzyme activities in rats but not in rabbits (Kiss et al., 1992
; Sastry et al., 1995
; Veltman et al.,1998). Moreover, expression of catalase, superoxide dismutase, and glutathione peroxidase is altered by hyperglycemic states (Reddi and Bollineni, 1997). Both catalase and superoxide dismutase are inducible (Shull et al., 1991
), and their activities are changed in diabetes (Kakkar et al., 1995
). Because aldose reductase inhibitors are structurally similar to some antioxidants, the question arises whether aldose reductase inhibitors might alter components of the oxidative stress pathway during normal levels of oxidative stress.
Therefore, this study has examined the hypothesis that treatment with aldose reductase inhibitors AL-1576 and AL-4114 influences the activities of enzymes (catalase, superoxide dismutase, glutathione reductase, and glutathione peroxidase) related to oxidative stress, the concentration of reduced glutathione (GSH) and glutathione disulfide (GSSG), and membrane lipid peroxidation in normal rat and rabbit livers.
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MATERIALS AND METHODS |
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Animals.
Male Sprague-Dawley rats (100130g) were purchased from Harlan Sprague Dawley (Indianapolis, IN), and pathogen-free New Zealand White rabbits (approximately 2 kg) came from Myrtle's Rabbitry (Nashville, TN). Both rats and rabbits were housed in stainless steel cages, and were provided Purina rat (#5012) or rabbit (#5326) chow and water ad libitum. Animal husbandry and experimentation were consistent with the Guiding Principles in the Use of Animals in Toxicology and the USPHS Guide for the Care and Use of Laboratory Animals. After a 4-day acclimation interval, rats and rabbits were each divided randomly into 5 groups of 8 animals. Treatment groups included: control (1.0% NaHCO3 vehicle), AL-1576 low dose (10 mg/kg) and high dose (50 mg/kg), and AL-4114 low dose (10 mg/kg) and high dose (50 mg/kg). The two doses are known (Kiss et al. 1992; Sastry et al., 1995
) to produce dose-dependent inhibition of aldose reductase. Each animal received a 1-ml (rat) or 2-ml (rabbit) injection ip once daily for 4 days. On day 5, rats were anesthetized with sodium pentobarbital (100 mg/kg); rabbits were sedated with ketamine (0.5 ml intramuscular) then euthanized with 1-ml pentobarbital by cardiac puncture. Livers were removed, rinsed in ice-cold 1.15 % KCl, and immediately frozen at 70°C. Previous experiments with liver indicated that all measured enzyme activities were stable to storage for several months at 70°C.
Tissue preparation.
Liver (500 mg of tissue/4.5ml of 0.1 M TrisHCl buffer, pH 7.4) was homogenized using a Brinkmann Polytron homogenizer set between 5 and 6. This 10% homogenate was centrifuged at 100,000 x g for one h, the supernatant (cytosols) carefully poured off, and the pellet discarded. The 10% cytosols were assayed for protein content (Lowry et al., 1951), allocated into separate Eppendorf tubes, and frozen for future enzyme assays.
Assays.
Lipid peroxidation was measured by determining the concentration of thiobarbituric acid reactive substances (Ohkawa et al., 1979) in fresh 10% liver homogenates. Concentrations of GSH and GSSG in liver were measured according to Hissin and Hilf (1976). Activities of catalase (Luck, 1963
), superoxide dismutase (Crapo et al., 1978
), glutathione peroxidase (Tappel, 1978
), and glutathione reductase (Carlberg and Mannervik, 1975
) were measured in 10% cytosols.
Statistics.
Means and standard errors of the data were analyzed by ANOVA and Duncan's test; p < 0.05 was considered significant. The graphs are presented as percent of control.
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RESULTS |
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DISCUSSION |
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Because AL-4114 and AL-1576 (Fig. 4) are structurally similar to many antioxidants found in plants, such as carnosol, rosmarinic acid, and curcumin (Halliwell, et al., 1995
), these two aldose reductase inhibitors may also function as antioxidants. An antioxidant can act by inhibiting generation of ROS, by directly scavenging free radicals, or by raising the levels of endogenous antioxidant defense. Structural analysis would suggest that the methoxy groups on AL-4114 would be better electron donors than the hydrogens on AL-1576, making AL-4114 the more effective antioxidant. Both aldose reductase inhibitors have been shown to attenuate development of diabetic cataracts (Reddy et al., 1992
), but AL-4114 was the most potent of six aldose reductase inhibitors in both human lens epithelium (HLE) and human retinal pigment epithelium (HRPE) (Reddy et al., 1992
). However, consistently higher doses of aldose reductase inhibitor were required to disrupt polyol formation in HRPE than in HLE. AL-1576 was equivalent to AL-4114 as an inhibitor in HRPE, but was less effective in the HLE. In our study, AL-4114 treatment caused more effects on the oxidative stress pathway in normal rabbit liver than did AL-1576, supporting the suggestion that AL-4114 may be a more effective antioxidant. The decreased activity of glutathione reductase in normal rabbit liver after treatment with high and low doses of AL-4114, and the decreased activity of superoxide dismutase and glutathione peroxidase in normal rabbit liver after treatment with low doses of AL-4114, do not appear to result in increased lipid peroxidation, perhaps because the antioxidant function of AL-4114 compensates for that of the enzymes.
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Oxidative stress, antioxidants, and the polyol pathway are known to be linked in pathological states; changes in the polyol pathway can change the redox status of cells, thereby altering the activities of oxidative enzymes. For instance, activation of the polyol pathway in hyperglycemia disrupts the NADP+/NADPH balance within the cell (Asahina et al., 1995), thereby altering the redox status, inhibiting other NADPH-requiring enzymes, and modifying the antioxidant pathway within tissues that have endogenous aldose reductase, including retina, kidney, and neurons (Baynes, 1991
; Chandler and Miller, 1986
; Ludvigson et al., 1980). This redox imbalance is also evident in pseudohypoxia, where the NADH/NAD+ ratio is increased in tissues with a normal partial pressure of oxygen (Williamson et al., 1993
). Clinically, while activation of the polyol pathway in non-insulin dependent diabetic patients decreased NADPH and GSH levels (Bravi et al., 1997
), treatment with an aldose reductase inhibitor restored the GSH level and the NADPH/NADP+ ratio to essentially normal values. Increased concentrations of ROS and decreased levels of endogenous antioxidants as seen by increased lipid peroxidation in poorly controlled diabetic patients (Altomare et al., 1997
, 1992
) are implicated in the etiology of diabetic complications. All of this evidence supports the argument that these diabetic complications, although they appear diverse and unrelated, may actually share common etiology via the oxidative stress pathway.
In conclusion, these aldose reductase inhibitors could become an important research tool that might lead to a deeper understanding of the relationship between the polyol and the oxidative stress pathways, both in normal animals and in those with pathological metabolic conditions such as diabetes. Aldose reductase inhibitors are known to decrease oxidative stress in diabetic patients. Therefore, this study set out to determine whether these compounds would alter hepatic antioxidant components in normal animals, and showed that aldose reductase inhibitors AL-1576 and AL- 4114 had no appreciable effects on antioxidant components in the normal liver, except for glutathione reductase.
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NOTES |
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