Dehydroepiandrosterone protects tissues of streptozotocin-treated rats against oxidative stress

Free Radical Biology & Medicine, Vol. 26, Nos. 11/12, pp. 1467–1474, 1999 Copyright © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/99/$–see front matter

PII S0891-5849(99)00012-X

Original Contribution DEHYDROEPIANDROSTERONE PROTECTS TISSUES OF STREPTOZOTOCINTREATED RATS AGAINST OXIDATIVE STRESS MANUELA ARAGNO,* ELENA TAMAGNO,* VALENTINA GATTO,† ENRICO BRIGNARDELLO,† SILVIA PAROLA,* OLIVIERO DANNI,* and GIUSEPPE BOCCUZZI† *Department of Experimental Medicine and Oncology, General Pathology Section, and †Department of Clinical Pathophysiology, University of Turin, Turin, Italy (Received 23 September 1998; Revised 2 December, 1998; Accepted 5 January 1999)

Abstract—Chronic hyperglycemia in diabetes determines the overproduction of free radicals, and evidence is increasing that these contribute to the development of diabetic complications. It has recently been reported that dehydroepiandrosterone possesses antioxidant properties; this study evaluates whether, administered daily for three weeks per os, it may provide antioxidant protection in tissues of rats with streptozotocin-induced diabetes. Lipid peroxidation was evaluated on liver, brain and kidney homogenates from diabetic animals, measuring both steady-state concentrations of thiobarbituric acid reactive substances and fluorescent chromolipids. Hyperglycemic rats had higher thiobarbituric acid reactive substances formation and fluorescent chromolipids levels than controls. Dehydroepiandrosterone-treatment (4 mg/day for 3 weeks) protected tissues against lipid peroxidation: liver, kidney and brain homogenates from dehydroepiandrosterone-treated animals showed a significant decrease of both thiobarbituric acid reactive substances and fluorescent chromolipids formation. The effect of dehydroepiandrosterone on the cellular antioxidant defenses was also investigated, as impaired antioxidant enzyme activities were considered proof of oxygen-dependent toxicity. In kidney and liver homogenates, dehydroepiandrosterone treatment restored to near-control values the cytosolic level of reduced glutathione, as well as the enzymatic activities of superoxidedismutase, glutathione-peroxidase, catalase. In the brain, only an increase of catalase activity was evident (p , .05), which reverted with dehydroepiandrosterone treatment. The results demonstrate that DHEA treatment clearly reduces oxidative stress products in the tissues of streptozotocin-treated rats. © 1999 Elsevier Science Inc. Keywords—Streptozotocin, Thiobarbituric acid reactive substances, Superoxide dismutase, Dehydroepiandrosterone, Oxidative stress, Free radicals

INTRODUCTION

Dehydroepiandrosterone (DHEA), the major secretory product of the human adrenal gland, has been reported to possess both pro-oxidant [13–15] and antioxidant properties [16 –21], probably depending on protocol and/or doses. We recently showed that DHEA exerts a multi-targeted antioxidant activity in vivo in rats [22] and prevents oxidative damage, whether induced by acute hyperglycemia [23] or in an in vitro model [24]. In the present study, we examine the conditions under which DHEA, given per os, provides antioxidant tissue protection in rats with STZ-induced diabetes. Both lipid peroxidation and cellular antioxidant defenses are investigated, the increase of antioxidant enzyme activity during hyperglycemia being considered proof of oxygendependent toxicity [25,26].

Chronic hyperglycemia is the primer of a series of cascade-reactions causing the overproduction of free radicals [1– 4], and increasing evidence indicates that these contribute to the development of diabetic complications. Several studies have shown that treatment with antioxidants reduces diabetic complications [5,6]: they attenuate the development of peripheral nerve dysfunction [7,8], normalize the endothelial function in diabetes [9,10] and exert beneficial effects on platelet hyperaggregability [11]. Moreover, antioxidants decrease the occurence of malformations in the offspring of diabetic rats [12]. Address correspondence to: Prof. Giuseppe Boccuzzi, Department of Clinical Pathophysiology, Via Genova 3, 10126 Torino, Italy; Tel: 139 (11) 663-1216; Fax: 139 (11) 667-0436; E-Mail: [email protected]. 1467

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Table 1. Level of Glucose, DHEA and Nonenzymatic Antioxidants in Plasma of Control and STZ-Treated Rats at Time of Death (3 Weeks After Start of DHEA or Vehicle Treatment). CONTROL

Glucose (mmol/l) DHEA (ng/ml) a-Tocopherol (nmol/ml) GSH (mmol/ml)

STZ

2DHEA

1DHEA

2DHEA

1DHEA

7.33 6 1.59 1.50 6 0.81 42.75 6 1.54 0.90 6 0.19

8.22 6 0.17 4.80 6 2.11 43.98 6 1.69 0.97 6 0.25

48.03 6 9.30* 0.51 6 0.30 22.44 6 3.40* 0.45 6 0.08*

45.50 6 7.06‡ 4.41 6 3.32 19.61 6 1.07‡ 0.68 6 0.11†

Values are means of four to seven rats per groups 6 S.D. Statistical significance: STZ vs. C: *p , .05. STZ 1 DHEA vs. STZ: †p , .05. STZ 1 DHEA vs STZ: ‡Nonsignificant MATERIALS AND METHODS

All reagents used were from Merck (Darmstadt, Germany) unless otherwise specified. Male Wistar rats (Harlan-Nossan, Correzzana, Italy) ranging between 200–220 g body weight (b.wt.) were housed and given human care in compliance with the Italian Ministry of Health Guidelines and with the Principles of Laboratory Animal Care (NIH n° 85–23, revised 1985). Animals were provided with Piccioni pellet-diet (n° 48, Gessate Milanese, Italy) and water ad libitum. One week after their arrival, the animals were randomly divided into two groups: the control group, receiving saline, and the streptozotocin (STZ)-group. Streptozotocin (Sigma Chemical Company, St. Louis, MO, USA) dissolved in citrate buffer 0.05 M pH 4.5, was injected via the tail vein, vehicled in 0.25 ml / 100 g b.wt of a freshly prepared solution, to give a final dose of 50 mg/kg b.wt. STZ was rapidly eliminated from the body: about 80% appeared in the urine within 6 h [3]. Hyperglycemia was confirmed on the third day postinjection (o-toluidine reagent, Sigma Chemical Company). Rats were removed from the study if the blood glucose level in the fed state was below 20 mmol/l. On the fourth day post injection, the hyperglycemic animals were randomly divided into two sub-groups: one group (n 5 6) received DHEA daily (4 mg in 0.5 ml of mineral oil per animal) and the other group (n 5 7) received an equal volume of vehicle. Animals were not treated with insulin at any time. The control rats were divided into two groups: one group (n 5 5) was treated with dehydroepiandrosterone daily (4 mg in 0.5 ml of mineral oil per animal) and the other (n 5 6) with an equal volume of mineral oil. Dehydroepiandrosterone or vehicle was administered by gastric gavage for 21 consecutive days at 0900. The following day the rats were killed: they were anaesthetized by halothane and a laparatomy was performed. Blood was drawn from the bifurcation of the aorta, collected in tubes containing ethylenediaminetetraacetate (EDTA) 5% (w:v). Plasma was separated by centrifugation and stored at 280°C. On a

portion of fresh plasma, the glucose concentration was analyzed. Liver, kidney and brain were removed, weighed, and divided into portions.

Biochemical determinations On fresh tissue, lipid peroxidation levels were determined in terms of the formation of thiobarbituric acid reactive substances (TBARS) [27] and of the production of fluorescent chromolipids [28]. Thiobarbituric acid reactive substances formation was evaluated in liver, kidney, and brain homogenates, prepared in 15 mM Na phosphate buffer, pH 7.4 and incubated in a shaking bath at 37°C for up to 180 min. Aliquots were taken at different times and mixed with 10% TCA (v: v). After centrifugation, portions of supernatant were mixed with thiobarbituric acid, and TBARS formation was spectrophotometrically measured at 543 nm. Fluorescent chromolipids were determined in 1% (w:v) liver, kidney, and brain homogenates. Total lipids were extracted by Folch’s method [29]. The fluorescence intensity of the samples was monitored at 360 nm excitation and 430 nm emission with an LS-5-Luminescence Spectrometer. Dehydroepiandrosterone concentration was measured in the plasma: samples were extracted with ethyl ether, evaporated, and the residue redissolved in 0.3 ml isooctaneethylacetate (94:4, v:v) and chromatographed on celite: ethyleneglycol (2:1,w:v) micro-columns, using isooctanebenzene (94:4, v:v) as mobile phase. RIA was performed on the resulting DHEA chromatographic fraction [30]. Reduced glutathione content was determined in tissue homogenates and plasma by the Owens and Belcher procedure [31]. Tissue homogenates (10%,w/v) prepared in TCA-EDTA (10%-10mM,v:v) were centrifuged and the supernatant used. A mixture was directly prepared in cuvette: 0.05 M Na-phosphate buffer, pH 7.0; 1 mM EDTA, pH 7.0; 10 mM DTNB and an aliquot of the sample was monitored at 412 nm for two min (total

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nates as described by Flohe´ and Otting [33]: a solution of 5 mmol of xanthine (Sigma) in 0.001 N sodium hydroxide and 2 mmol of cytochrome C (Sigma) was mixed with 50 mM phosphate buffer pH 7.8 containing 0.1 mM EDTA. Since the activity of xanthine-oxidase (Sigma) may vary, sufficient enzyme should be used to produce a rate of cytochrome C reduction of at least 0.025 absorbance units/min in the assay without SOD. Isolation of cytosolic fraction Additional controls and animals treated with STZ, with or without DHEA, were anaesthetized by halothane and killed by aorta bleeding, after which cold NaCl 0.9% saline was used for perfusion. Liver, kidney, and brain were removed and weighed. Tissues were homogenated in KCl 1.15% (40%, w/v) by Potter-Elvehjem. The suspensions were centrifuged once at 15,000 g at 4°C for 18 min, then again at 105,000 g at 4°C for 40 min. Cytosolic fractions of the three tissues were stored at 280°C. Portions of cytosol of liver, kidney, and brain were utilized to measure: a) glutathione reductase (GRD) activity, by the Hosoda and Nakamura method [34]; b) glutathione peroxidase (GPX) activity, using hydrogen peroxide as substrate for Se-dependent activity and terzbutylhydroperoxide for Se-independent activity [35]; c) catalase activity, by Aebi’s method [36]. Cytosolic proteins of different tissues were determined by Lowry’s method [37]. Aldose reductase activity was determined in kidney and brain cytosol by the Wermhuth and Von Wartburg method [38]. The substrate specificity of aldose reductase was determined by kinetic analysis at 340 nm in 0.1M sodium phosphate, pH 7.0, containing 0.1 mM d-xylose and 0.01M diphenylhydantoin dissolved in 0.01M NaOH. Fig. 1. Fluorescent chromolipids in liver (A), kidney (B), and brain (C) homogenates of control and STZ-treated rats with or without DHEA. Number of rats in brackets. Means 6 standard deviation. Statistical significance: STZ vs. C: * p , .05; STZ 1 DHEA vs. STZ: † p , 0.05.

thiol), later a suitable diluted glutathione reductase and NADPH were added (total glutathione). a-Tocopherol was assayed on tissue homogenates and plasma by the method described by Burton et al. [32]. After extraction with 1 ml n-heptane and brief centrifugation, the heptane phase was collected for HPLC analysis; a Supercosil-plc-si column (25 cm * 4.6 m, Supelco Inc., PA, USA) was used; the mobile phase was hexaneisopropanol (99:1, v:v) and the flow rate 1.5 ml/min; the fluorescence detector was set to 298 nm excitation and 325 nm emission. Total SOD activity was assayed in tissue homoge-

Statistical analysis of data Values are means of between four and seven animals per group 6 standard deviation. Differences between means were analyzed for significance using one-way ANOVA test, performed with the Bonferroni post test [39]. RESULTS

Diabetes was induced in rats by a single injection of STZ. Dehydroepiandrosterone treatment started on day four after STZ-injection. Table 1 shows plasma levels of DHEA, glucose, and primary endogenous and exogenous antioxidants, after 21 days of DHEA treatment. Glucose in STZ rats rapidly increased and was not modified by DHEA treatment. During DHEA treatment, the plasma

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level of the steroid reached values similar to that normal for an elderly man [40]. The level of a-tocopherol decreased in the diabetic rats and was unchanged by DHEA treatment. Conversely, the decrease of glutathione content, observed in diabetic rats, was significantly (p , .05) attenuated by treatment with the steroid. Lipid peroxidation in tissues There was no variation in the absolute mean of wet weights of liver, kidney, and brain in normal and STZ-rats, treated and untreated with DHEA. Diabetic rats lost body weight during the experiment (about 25%). DHEA treatment partially prevented the body weight loss induced by diabetes (data not shown). Fig. 1 shows the fluorescent chromolipid levels in control and STZ rats, with or without DHEA. Homogenates of liver (A), kidney (B) and brain (C) prepared from tissues of diabetic rats showed a marked increase of fluorescent chromolipids levels, indicating the presence of oxidative damage induced by hyperglycemia. Dehydroepiandrosterone treatment significantly (p , .05) reduced fluorescent chromolipids. TBARS production was higher in the homogenates of diabetic rats incubated for up to 240 min than it was in controls (Fig. 2). DHEA-treatment gave a significant (p , .05) reduction of TBARS formation, indicating that the tissue oxidative status markedly improved with DHEA treatment. Nonenzymatic defenses of tissues against free radicals The content of both GSH and alpha-tocopherol were reduced in liver and kidney homogenates of STZ rats, as documented previously [25,41]. Dehydroepiandrosterone treatment in STZ rats was able to prevent GSH loss in tissues: GSH level of DHEA-treated diabetic rats did not differ from controls (Tables 2 and 3). Conversely, DHEA treatment did not modify the level of exogenous nonenzymatic antioxidant a-tocopherol (Tables 2 and 3). In the brain tissue, Table 4, the level of tripeptide was not changed by STZ treatment, while a-tocopherol showed a marked decrease after STZ treatment; this decrease was not reverted by DHEA treatment. Enzymatic defenses of tissues against free radicals The general patterns of enzymatic antioxidants in diabetic rats are reported in Tables 2– 4. The total SOD

Fig. 2. TBARS formation in liver (A), kidney (B) and brain (C) homogenates of control and STZ-treated rats with or without DHEA. Control ( E ), DHEA ( h ), STZ (F), STZ1DHEA (■). Values are means of between four to seven animals per group 6 standard deviation. Statistical significance: STZ vs C: * p , .05; STZ1DHEA vs STZ: † p , .05.

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Table 2. Levels of Nonenzymatic and Enzymatic Antioxidants in Liver of Control and STZ-Treated Rats, With or Without DHEA Control

GSH (nmol/mg prot) a-Tocopherol (pmol/mg prot) Total-SOD (U/min/mg prot) GPX Sel dep. (nmol/min/mg prot) GRD (nmol/min/mg prot) Catalase (pmol/mg prot)

STZ

2DHEA

1DHEA

2DHEA

1DHEA

66.18 6 8.08 233.03 6 42.75 283.31 6 46.58 223.14 6 42.13 55.55 6 13.26 118.50 6 16.40

64.19 6 10.39 241.74 6 42.30 253.53 6 60.13 216.78 6 43.13 60.31 6 18.84 118.45 6 12.84

45.70 6 7.45* 141.06 6 34.10* 167.19 6 52.52* 415.98 6 32.64* 60.31 6 20.46 189.17 6 25.42*

63.33 6 7.95† 138.41 6 45.88‡ 260.67 6 48.13† 248.37 6 34.17† 60.84 6 19.44 131.25 6 17.01†

Values are means of four to seven rats per groups 6 standard deviation. Statistical significance: STZ vs. C: *p , .05. STZ 1 DHEA vs. STZ: †p , .05. STZ 1 DHEA vs. STZ: ‡Nonsignificant.

activity decreased in the liver tissue of diabetic rats. DHEA-treatment restored SOD activity to control values (Table 2). Liver and kidney tissues of diabetic rats showed a marked increase in GPX-Se-dep and catalase activities. DHEA treatment was found to normalize GPX Se-dep and catalase activities in both tissues (Tables 2 and 3). GPX Se-independent activity showed a similar pattern to GPX Se-dep (data not shown). Catalase activity was increased in brain tissue of STZ rats, whereas GPX-Se-dep activity was unchanged. DHEA treatment brought catalase activity to normal values. GRD activity was not significantly different from that of the control rats in any of the conditions studied (Tables 2– 4). Finally, Table 5 shows aldose reductase activity in kidney and brain cytosol. STZ produced a significant increase of this activity with respect to controls (p , 0.05), partially prevented by DHEA treatment. The enzyme is not present in the liver [38]. DISCUSSION

The increase of free-radical mediated-toxicity, spread throughout different tissues, is well documented in clinical diabetes [42,43] and in STZ-diabetic rats [5]. These studies show that DHEA administered daily for three

weeks to STZ rats has a protective effect against the oxidative stress induced by chronic hyperglycemia. The peroxidative damage to liver, kidney, and brain is demonstrated by the high tissue content of fluorescent chromolipids, formed from peroxidative damage of polyunsaturated fatty acids, and by the tissues’ enhanced susceptibility to lipid peroxidation, in terms of TBARS production. When DHEA was administered to STZ rats, the oxidative status of the tissues was less expressed. STZ is rapidly eliminated from the body [3]; thus the reported effect of DHEA on STZ-treated rats cannot be attributed to interference between DHEA and STZ [25, 44]. We have shown, in animal models, that DHEA treatment protects the liver against oxidative damage [17,18], as well as protecting low-density lipoprotein (LDL) in the plasma and brain microsomes [22], indicating the effectiveness and multi-targeted antioxidant activity of DHEA. DHEA treatment has also been seen to exert a protective effect against lipid peroxidation triggered by acute hyperglycemia [23]. We show here that the effectiveness and multi-targeted antioxidant effect of DHEA is maintained during chronic treatment, when DHEA plasma levels reached values similar to those found in man [40]. The mechanism underlying the DHEA-induced lipid protection against oxidation remains to be defined. It has been proposed that DHEA

Table 3. Levels of Nonenzymatic and Enzymatic Antioxidants in Kidney of Control and STZ-Treated Rats, With or Without DHEA. Control

GSH (nmol/mg prot) a-Tocopherol (pmol/mg prot) Total-SOD (U/min/mg prot) GPX Sel dep. (nmol/min/mg prot) GRD (nmol/min/mg prot) Catalase (pmol/mg prot)

STZ

2DHEA

1DHEA

2DHEA

1DHEA

21.82 6 4.30 196.35 6 39.83 259.89 6 50.58 135.22 6 44.10 63.08 6 12.89 105.91 6 17.72

21.74 6 2.75 233.53 6 49.61 255.88 6 68.88 139.98 6 38.10 59.69 6 17.06 116.81 6 23.60

9.89 6 2.50* 108.65 6 29.15* 219.28 6 44.78 255.30 6 40.88* 78.13 6 16.74 184.09 6 21.36*

17.36 6 5.50† 98.55 6 19.52‡ 239.32 6 41.43 148.76 6 44.32† 72.57 6 11.09 125.10 6 21.11†

Values are means of four to seven rats per groups 6 S.D. Statistical significance: STZ vs. C: *p , 0.05. STZ 1 DHEA vs. STZ: †p , 0.05. STZ 1 DHEA vs. STZ: ‡Nonsignificant.

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M. ARAGNO et al. Table 4. Levels of Nonenzymatic and Enzymatic Antioxidants in Brain of Control and STZ-Treated Rats, With or Without DHEA. Control

GSH (nmol/mg prot) a-Tocopherol (pmol/mg prot) total-SOD (U/min/mg prot) GPX Sel dep. (nmol/min/mg prot) GRD (nmol/min/mg prot) Catalase (pmol/mg prot)

STZ

2DHEA

1DHEA

2DHEA

1DHEA

19.20 6 1.63 193.45 6 35.20 327.02 6 64.29 84.22 6 28.10 69.06 6 6.73 34.44 6 4.44

18.89 6 3.21 211.30 6 31.50 333.07 6 47.27 72.98 6 22.10 61.79 6 4.22 33.87 6 7.08

17.72 6 2.30 103.80 6 29.11* 245.25 6 49.54 112.30 6 25.88 69.31 6 13.34 52.25 6 6.61*

17.55 6 1.82 96.55 6 26.68‡ 315.87 6 51.04 78.76 6 31.32 76.23 6 12.24 31.11 6 4.22†

Values are means of four to seven rats per groups 6 standard deviation. Statistical significance: STZ vs. C: *p , .05. STZ 1 DHEA vs. STZ: †p , .05. STZ 1 DHEA vs. STZ: ‡Nonsignificant.

nonenzymatic antioxidants (the exogenous a-tocopherol and the endogenous GSH) are reduced by 50% in the liver and kidney tissues of diabetic rats, whereas the detoxifying enzymes are induced: both GSH-dependent enzymes and catalase were significantly increased in the liver and kidney tissues analyzed. The existence of oxidative damage in STZ rats is also confirmed by the altered pattern of SOD. Superoxide-dismutase activity shows both an increase and a reduction in experimental diabetes, partially related to both duration and severity of the disease [3,55]. We observe a significant decrease of SOD activity in the liver of STZ rats, in agreement with findings reported by others [25]. Reactive oxygen radicals could themselves reduce SOD activity [56]. In addition, this decrease might depend on the changes in glutathione levels, since GSH promotes SOD synthesis [41]. SOD activity was unchanged in the brain of STZtreated rats, in agreement with previous reports [57], whereas catalase activity was increased. Because H2O2 is a highly lipid soluble oxygen species [51], it might diffuse in brain tissue better than the other oxygen radicals, including O2•2 , which might explain the different brain tissue response to oxidative stress. Here we show that, when DHEA is administered to STZ rats, the pattern of both nonenzymatic and enzymatic antioxidants reverts to near-control values. Only a-tocopherol, being exogenous, remains low: its level cannot be reverted

activity requires its transformation [45]. Indeed, most of the effects attributed to DHEA are mediated by one or another of its metabolites, such as 5-en-androsten-3b, 17b-diol for the hormonal effect [46] or 5-en-androsten-3b,7b,17b-triol for the action on the immune system [47]. Whichever the active compound is, one might hypothesize that it modifies the membrane lipid composition or structure, in analogy to the stable modifications of lipoprotein induced by estradiol [48], making them more resistant to attack by free radicals. It has been reported that DHEA is able to change the fatty acid composition of mitochondria phospholipid membrane in rats [49]. Moreover, plasma LDL are less prone to peroxidation when incubated with DHEA [22,50]. Alternatively, DHEA might counteract the delocalization of iron from its binding sites, induced by diabetic acidosis. This might protect tissue from the iron catalyzed oxygen freeradical production [51]. This hypothesis is supported by the effect of steroids on the iron redox cycle [52], but it would not explain why DHEA antioxidant activity has also been observed in iron-independent experimental models [17,22]. It may also be relevant that DHEA inhibits glucose-6phosphate dehydrogenase (G6PDH) activity and thereby reduces NADPH, but at DHEA concentrations higher than those reached in our experiments [53]. Here we confirm the previous finding that the increase of peroxidative processes in tissue generates a strong imbalance of cellular defense systems [54]. Levels of

Table 5. Aldose Reductase Activity in Kidney and Brain of Control and STZ-Treated Rats, With or Without DHEA. CONTROL

Kidney (nmol/min/mg prot) Brain (nmol/min/mg prot)

STZ

2DHEA

1DHEA

2DHEA

1DHEA

3.42 6 0.28 1.12 6 0.20

3.70 6 0.49 1.16 6 0.13

4.70 6 0.37* 1.90 6 0.17*

3.92 6 0.51†§ 1.41 6 0.18†§

Values are means of four to seven rats per groups 6 standard deviation. Statistical significance: STZ vs. C: *p , .05. STZ 1 DHEA vs. STZ: †p , .05. STZ 1 DHEA vs. C: ‡p , .05.

Dehydroepiandrosterone and oxidative stress

to normal value without dietary supplementation. Reduced glutathione, GSH-dependent enzymes, SOD, and catalase return to near-control values. GSH level correction might be crucial in protecting against the development of some diabetic complications. Besides its role in many biological processes, such as protein and nucleic acid synthesis, GSH reduces disulphide linkage of proteins and other cellular molecules involved in tissue damage [58]. In experimental diabetes, the increase of sorbitol levels, caused by increased aldose reductase activity, also damages the tissues. Sorbitol plays a major role in diabetic neuropathy [59]. We show that the increase of aldose reductase activity in kidney and brain tissues of hyperglycemic rats is still evident in animals treated with DHEA, although to a lesser extent, which confirms the generally held opinion that aldose reductase is induced in hyperglycemia by osmotic and glucose response elements [60,61]. The new information is that hyperglycemic rats treated with DHEA show a reduced activity of aldose reductase, which can be ascribed to the decrease of final products of lipid peroxidation: in fact, the aldose reductase has also been shown to be induced by the aldehyde derivate of lipid oxidation [62]. However, it remains to be demonstrated that the decrease in aldose reductase activity observed in DHEA-treated STZ rats is sufficient to affect tissue sorbitol concentrations. In conclusion, peroxidative processes which are reported to be involved in diabetic complications might be counteracted by DHEA administration: besides the reduction of lipid membrane susceptibility to oxidation, DHEA also prevents the change of both enzymatic and non-enzymatic antioxidants induced by oxidative stress. It is notable that the antioxidant effect of steroid in hyperglycemic rats occurs when the DHEA plasma level is close to normal human values. The protective effect of DHEA, a steroid whose production rate varies widely among individuals within a normal population [63], should be taken into account when speculating about differences in susceptibility to free radical–mediated tissue damage among patients with similar duration and metabolic control of diabetes. Acknowledgements — This study was supported by the Ministero dell’Universita` e della Ricerca Scientifica e Tecnologica (60 and 40%, respectively).

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SOD—superoxide-dismutase GSH—reduced glutathione DHEA— dehydroepiandrosterone STZ—streptozotocin TBARS—thiobarbituric acid reactive substances