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Melatonin attenuates diabetes-induced oxidative stress in rabbits
Introduction
It is commonly accepted that oxidative stress plays a crucialrole in the development of diabetic complications [1, 2].
However, the present approaches to diabetes therapyinvolve mainly drugs enhancing insulin secretion or signal-ling as well as inhibitors of endogenous glucose production
[cf. 3], underestimating the role of antioxidants as agentsimportant for restoring the redox balance of the organism.Moreover, some compounds that seem promising in termsof the attenuation of diabetic hyperglycaemia, e.g. tung-
state and vanadyl acetylacetonate, appear to exhibit a pro-oxidative action [4, 5], so the combined therapy withhypoglycaemic compounds and antioxidants needs a care-
ful evaluation.Melatonin, which is produced both in the pineal gland
and gastrointestinal tract, is one of the compounds most
extensively studied in view of its potent antioxidativeactivity [6]. Similarly, N-acetylcysteine (NAC) is not onlya precursor of cysteine, the amino acid limiting the rate of
the biosynthesis of glutathione, which is considered to bethe main thiol antioxidant in mammalian cells [7], but alsoseems to exhibit direct antioxidative action as a free radicalscavenger [8, 9]. However, the knowledge concerning the
effects of these compounds on hyperglycaemia-inducedoxidative stress is still incomplete.
The aim of this study was to establish whether melatoninor NAC might be beneficial for diabetes therapy. In order
to determine oxidative stress intensity, hydroxyl free radical(HFR) levels and glutathione redox state were measured inserum, liver and kidney cortex of diabetic rabbits both
untreated and treated with either melatonin or NAC. Theactivities of the enzymes of glutathione metabolism, i.e.c-glutamylcysteine synthetase, glutathione reductase and
glutathione peroxidase have also been estimated in liver andrenal cortex of these animals.
Materials and methods
Animals
The experiments were performed with male Termondrabbits weighing approximately 2–2.5 kg. Animals weremaintained on standard rabbit chow with free access to
water and food. A group of eight control rabbits wasinjected with 1 mL of sterile citrate buffer (pH 4.5). Theremaining rabbits were made diabetic by a single intraven-ous injection of alloxan (175 mg/kg body weight) freshly
dissolved in 1 mL of sterile citrate buffer (pH 4.5) [10]. Onlythe alloxan-treated animals which exhibited decreased orstabilized body weight and blood glucose concentration in
excess of 300 mg/dL 3 days after the treatment were
Abstract: Oxidative stress is considered to be the main cause of diabetic
complications. As the role of antioxidants in diabetes therapy is still
underestimated, the aim of the present investigation was to study the
antioxidative action of melatonin in comparison with N-acetylcysteine
(NAC) under diabetic conditions. Alloxan-diabetic rabbits were treated daily
with either melatonin (1 mg/kg, i.p.), NAC (10 mg/kg, i.p.) or saline. Blood
glutathione redox state and serum hydroxyl free radicals (HFR), creatinine
and urea levels were monitored. After 3 wk of treatment animals were killed
and HFR content, reduced glutathione/oxidized glutathione (GSH/GSSG)
ratio as well as the activities of glutathione reductase, glutathione peroxidase
and c-glutamylcysteine synthetase were estimated in both liver and kidney
cortex. Diabetes evoked a several-fold increase in HFR levels accompanied
by a significant decline in GSH/GSSG ratio in serum and the examined
organs. In contrast to NAC, melatonin (at 1/10 the dose of NAC) attenuated
diabetes-induced alterations in glutathione redox state and HFR levels,
normalized creatinine concentration and diminished urea content in serum.
Moreover, the indole resulted in an increase in glutathione reductase activity
in both studied organs and in a rise in glutathione peroxidase and
c-glutamylcysteine synthetase activities in the liver. In contrast to NAC,
melatonin seems to be beneficial for diabetes therapy because of its potent
antioxidative and nephroprotective action. The indole-induced increase in
the activities of the enzymes of glutathione metabolism might be of
importance for antioxidative action of melatonin under diabetic conditions.
Katarzyna Winiarska, TomaszFraczyk, Dominika Malinska,Jakub Drozak and Jadwiga Bryla
Department of Metabolic Regulation, Institute
of Biochemistry, Warsaw University, Warsaw,
Poland
Key words: diabetes, glutathione metabolism,
melatonin, N-acetylcysteine, oxidative stress
Address reprint requests to Jadwiga Bryla,
PhD, Department of Metabolic Regulation,
Institute of Biochemistry, Warsaw University, I.
Miecznikowa 1, 02-096 Warsaw, Poland.
E-mail: [email protected]
Received July 25, 2005;
Accepted October 11, 2005.
J. Pineal Res. 2006; 40:168–176Doi:10.1111/j.1600-079X.2005.00295.x
� 2005 The AuthorsJournal compilation � 2005 Blackwell Munksgaard
Journal of Pineal Research
168
considered diabetic and used for experiments. Diabeticrabbits were divided into three groups of eight animals:(i) untreated, (ii) treated with melatonin, and (ii) treated
with NAC. Both melatonin (1 mg/kg body weight [11]) andNAC (10 mg/kg body weight [12]) were dissolved in 1 mLof sterile saline and applied intraperitoneally, daily for3 wk. The remaining diabetic rabbits received intraperito-
neal injections of saline. In each group, three rabbits wereintended for serum HFR levels determinations and 2 hrprior to sample withdrawal received intraperitoneal injec-
tions of sodium salicylate (SAL) in sterile saline (75 mg/kgbody weight) [13]. Salicylate-treated rabbits exhibited nochanges in serum glucose, creatinine and urea concentra-
tions as well as in blood glutathione content and redoxstate, comparing with animals that had not receivedsalicylate injections. After 3 wk of the experiment animalswere killed by intravenous injection of pentobarbital
(30 mg/kg). All animal treatment procedures wereapproved by the First Warsaw Local Commission for theEthics of Experimentation on Animals.
Sample preparation
Blood was withdrawn from marginal vein of the ear andcollected into heparinized tubules placed on ice. Quantitiesof 200 lL of whole blood were left for reduced glutathione
(GSH) and oxidized glutathione (GSSG) determinations,while the rest was centrifuged in order to separate bloodcells. The supernatants for HFR estimations were depro-teinized with 35% perchloric acid containing 1 mm ethy-
lenediaminetetraacetic acid (EDTA) and 4 mm sodiummetabisulfite (10:1), while glucose, urea and creatinine weredetermined in samples deproteinized with 1% Na2WO4 in
30 mm H2SO4 (1:6).Blood samples for glutathione measurements were proc-
essed according to Dincer et al. [14]. Quantities of 100 lLof blood were added to 2.4 mL of the precipitating solutioncontaining 0.13 m metaphosphoric acid, 4 mm EDTA and3.2 m NaCl and thoroughly mixed. In case of GSSGdetermination 50 mm N-ethylmaleimide was added to the
mixture in order to avoid nonenzymatic GSH oxidation[15]. The samples were left for 5 min at room temperatureand then centrifuged. The supernatants were used for
glutathione determinations, following the removal of theexcess of N-ethylmaleimide by hexane extraction.
Kidney cortex and liver homogenates for HFR deter-
mination were prepared by the modified method ofMcCabe et al. [13]. Cortex tissue was homogenized in ice-cold 0.9% NaCl (2 g per 5 mL). The homogenate was
mixed with the equal volume of 0.4 m perchloric acidcontaining 200 lm EDTA and 200 lm sodium metabisulfiteand then sonicated. HFR levels were measured in thesupernatant obtained after the centrifugation of the hom-
ogenate.Kidneys and livers for glutathione redox state measure-
ments were homogenized in ice-cold 0.9% NaCl (0.5 g per
10 mL and 0.5 g per 2 mL for GSH and GSSG determi-nations respectively). The homogenates intended for GSHdetermination were immediately mixed with the equal
volume of 24% perchloric acid, while samples for GSSGmeasurements were treated with perchloric acid enriched
with 100 mm N-ethylmaleimide. The supernatants obtainedafter centrifugation of the samples were used for glutathi-one determinations, following the removal of the excess of
N-ethylmaleimide by hexane extraction. Cytosol fractionsfor enzyme activity measurements were prepared as des-cribed by McLellan et al. [16].
Enzyme activities measurements
Glutathione reductase activity was determined according to
Bergmeyer [17], while glutathione peroxidase activity wasmeasured by the method of Paglia and Valentine [18].c-Glutamylcysteine synthetase activity was determined by
monitoring the rate of ATP consumption, as described byHamilton et al. [19] and Kim et al. [20].
Determination of hydroxyl free radicals
Hydroxyl free radicals were estimated as 2,3-dihydroxy-benzoic acid (2,3-DHBA) generated in the presence of SAL
[13] administered to rabbits as described in Animals section.To exclude salicylate concentration-dependent differencesin 2,3-DHBA formation during the 3 wk of the experiment,
serum HFR levels are expressed as 2,3-DHBA/SAL ratio[13, 21].2,3-Dihydroxybenzoic acid assays were performed by
high-performance liquid chromatography (HPLC) usingBeckman Ultrasphere ODS column (Beckman Instru-ments, Inc., San Ramon, CA, USA). The mobile phaseconsisted of 50 mm NaH2PO4, 1.125 mm sodium octane-
sulphonic acid, 0.2 mm EDTA, 3% methanol and 5.5%acetonitryl (v/v). pH was adjusted to 2.8 with 1 m
ortophosphoric acid. 110B System Gold HPLC (Beckman
Instruments) was equipped with Bio-Rad 1640 electro-chemical detector (Bio-Rad, Hercules, CA, USA) and aglassy carbon working electrode operating at +0.75 V
against Ag/AgCl reference electrode and detection rangeof 2 nA. The flow rate was 1 mL/min and all separationswere performed at 30�C. In order to determine SAL themethod was modified in the following way: acetonitryl
concentration in the mobile phase was 9.5%, the potentialon the working electrode and the detection range were+1 V and 20 nA respectively. Quantification was
achieved using external standards of 2,3-DHBA andSAL. Data from the detector were collected and integra-ted by PC equipped with appropriate interface and
chromatography software.
Other analytical methods
Glucose serum concentration was analysed with hexokin-ase and glucose-6-phosphate dehydrogenase [17], whileurea was measured as ammonium following sample
treatment with urease [22]. Creatinine was determined byJaffe’s reaction as described by Michalik et al. [23]. GSSGwas estimated fluorimetrically with glutathione reductase
[17]. GSH levels were determined by HPLC (BeckmanInstruments) following derivatization with N-(1-pyre-nyl)maleimide [24]. Protein content in cytosolic fractions
was evaluated spectrophotometrically according to Layne[25].
Melatonin in diabetes treatment
169
Chemicals
Enzymes, coenzymes and nucleotides for metabolite deter-
minations were purchased from Roche (Mannheim,Germany). All other chemicals were from Sigma Chemicals(St Louis, MO, USA).
Expression of results
The significance of the differences was estimated using
ANOVA. Values are expressed as mean ± S.E.M. for 3–5separate experiments.
Results
As shown in Fig. 1, 3 days after the induction of diabetes,i.e. on the first day of the experiment, serum HFR levels
were thrice higher than in control animals. During pro-longed diabetes HFR progressively accumulated in serumand during the third week of the experiment their content
was sevenfold higher than the control value.The administration of melatonin to diabetic rabbits
effectively lowered serum HFR levels. Following 2 wk of
melatonin treatment HFR content reached the value similarto that observed in serum of control rabbits, while NACadministration had a negligible effect on serum HFR level
of diabetic rabbits.Fig. 2 presents changes in blood GSH and GSSG
contents as well as in GSH/GSSG ratio during 3 wk ofdiabetes. It is interesting that the induction of diabetes
caused a 40% increase in blood GSH level. However, underthese conditions GSSG level was elevated by 30%, leadingto no significant changes in GSH/GSSG ratio. During
prolonged diabetes GSH level continually decreased, reach-ing a value far below the control one. Moreover, aprogressive rise in blood GSSG content was also observed.
As a consequence, following 2 wk of diabetes blood GSH/GSSG ratio stabilized at the level lower by 50% than thecontrol value.
Melatonin treatment restored the control blood gluta-thione redox state mainly due to maintaining GSH contentat the elevated level observed soon after the induction of
diabetes. Surprisingly, NAC administration did not affectblood glutathione content. However, a slight delay in bothGSSG accumulation and GSH/GSSG ratio decrease wasobserved in blood of diabetic rabbits that had received
NAC injections.Neither melatonin nor NAC ameliorated diabetic hyper-
glycaemia (data not shown). NAC had no effect on serum
creatinine and urea concentrations in diabetic rabbits(Fig. 3). However, it is worth noticing that the normaliza-tion of serum creatinine concentration was achieved
following a few days of melatonin administration todiabetic animals. Moreover, during the second week ofmelatonin treatment a slight decline in serum urea concen-tration was observed. Even though urea level still markedly
exceeded the control value, diminished serum concentra-tions of both creatinine and urea might provide someevidence of nephroprotective action of melatonin in
diabetic rabbits.As shown in Table 1, following 3 wk of diabetes, HFR
levels in renal cortex and liver increased ten- and sixfold
respectively. This effect was partially attenuated by mela-tonin administration, whereas NAC treatment was ineffec-tive.
Diabetes did not change GSH content of kidney cortexor of the liver. However, under prolonged diabetic condi-tions, GSH/GSSG ratios were diminished by about 30% asa result of an increase in GSSG level. Melatonin admin-
istration to diabetic animals restored control values of renalGSSG content and GSH/GSSG ratio. Similarly, in liver ofmelatonin-treated diabetic rabbits GSH/GSSG ratio was
also equal to that in liver of control animals, but it was dueto 45% elevation of GSH content under conditions ofunaltered GSSG level. Surprisingly, NAC administration to
diabetic rabbits improved neither renal nor liver glutathi-one redox status.
bb
b
bb
b b
b
b
a,b
a aa
a a a
ba,b
a,ba,b
0
1
2
3
(2,3
-DH
BA
)/(S
AL
) ×
10 -3
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
(Days)
Fig. 1. Serum hydroxyl free radical (HFR) levels of diabetic rabbits untreated ( ) and treated with either melatonin ( ) or N-acetylcysteine(NAC) ( ). All animals received sodium salicylate injections 2 hr prior to blood withdrawal. Melatonin, NAC and salicylate wereadministered as described in Materials and methods. Values are mean ± S.E.M. for three animals. Dotted line depicts 2,3-dihydroxy-benzoic acid/sodium salicylate (2,3-DHBA/SAL) ratio value for control rabbits equal to 0.380 · 10)3 ± 0.011 · 10)3. aP < 0.05 versusvalues for untreated diabetic rabbits, bP < 0.05 versus values for control animals.
Winiarska et al.
170
To elucidate the mechanism of melatonin-induced chan-ges in glutathione redox state, the effect of this antioxidant
on the activities of c-glutamylcysteine synthetase, glutathi-one reductase and glutathione peroxidase was studied bothin renal cortex and in liver. As presented in Table 2, the
activities of these enzymes were not changed under diabeticconditions. It is worth noticing, however, that melatoninadministration to diabetic rabbits resulted in a significant(about 40%) increase in both renal and liver glutathione
reductase activity. Moreover, following melatonin treat-ment, hepatic activities of c-glutamylcysteine synthetaseand glutathione peroxidase were also markedly elevated (by
30% and 150% respectively).
Discussion
The results presented in this paper are in agreement withthe common view that diabetes is accompanied by oxidative
stress, which is regarded as the main cause of diabeticcomplications. According to our findings, during prolongeddiabetes HFR levels progressively rise and, after 3 wk, theyexceeded by several-fold the control values in serum, kidney
cortex and liver (cf. Fig. 1 and Table 1 respectively).Similarly, an increase in reactive oxygen species concentra-tions in the presence of high glucose concentrations have
been observed in vitro in rat kidney medulla [26] and humanrenal tubules [27]. Moreover, accelerated production ofreactive oxygen species has been determined in vivo both in
streptozotocin-diabetic rats [28, 29] and in patients withtype 1 [30, 31] and type 2 [32] diabetes. It also should be
pointed out that increased lipid peroxidation is anotherphenomenon typical of both types of diabetes [33–35].
Diabetes-induced oxidative stress is manifested notonly by elevated reactive oxygen species or increasedmalondialdehyde levels but also by impaired antioxidative
defence, as concluded from disturbed glutathione home-ostasis (cf. Fig. 2, Table 1). Similar data, indicating adiminished GSH concentration in blood of rabbits with2-month alloxan diabetes, have been presented by Meral
et al. [36]. Moreover, a decreased GSH content has beenobserved in erythrocytes of either streptozotocin-diabeticrats [37, 38] or humans with both types of diabetes [33,
39]. Diabetes-evoked decline in GSH/GSSG ratio hasalso been reported for blood [40] and granulocytes [41] ofpatients with type 2 diabetes. It is intriguing, however,
that the onset of diabetes results in a short-term increasein total glutathione blood content (cf. Fig. 2). Similarobservations have been made by Gupta et al. [42], who
have found elevated contents of both GSH and GSSG inrat erythrocytes 7 days after diabetes induction. Thisphenomenon might be one of the mechanisms counter-acting diabetes-evoked oxidative stress. It is worth
noticing that the expression of c-glutamylcysteine synthe-tase, the key enzyme of glutathione biosynthesis, isstimulated by oxidative stress [43]. However, c-glut-amylcysteine synthetase expression is activated by insulin[44] and it cannot be excluded that a massive release ofthis hormone accompanying alloxan-evoked b-cells dam-
age might also affect the rate of glutathione productionduring the first days of diabetes.
b
b
b
b bb b
b
bb
b
a,ba,b a,b
a,ba,ba,b
0
200
400
600
800
(µM
)
GSSG
GSH
bbbbb
b
b
bbbbbbb
bbbbbb
b
0
20
40
60
80
(µM
)
GSH/GSSG
bbbb
bb
b
a,ba,ba,b
a aa a a
0
4
8
12
16
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
(Days)
Fig. 2. Blood reduced glutathione (GSH)and oxidized glutathione (GSSG) levels aswell as GSH/GSSG ratios of diabeticrabbits untreated ( ) and treated witheither melatonin ( ) or N-acetylcysteine(NAC) ( ). Melatonin and NAC wereadministered as described in Materialsand methods. Values are mean ± S.E.M.for five animals. Dotted lines depictvalues for control rabbits equal to349.8 ± 19.1 lm, 33.6 ± 2.7 lm and12.8 ± 0.6 for GSH level, GSSG contentand GSH/GSSG ratio respectively.aP < 0.05 versus values for untreateddiabetic rabbits, bP < 0.05 versus valuesfor control animals.
Melatonin in diabetes treatment
171
b
b
bbbbb
b
bb
b
bb
b
aaaaaaa
a,b
0
1
2
3
4
5
6
(mg
/dL
)
Urea
b
b
bb
bb
bbb
b
bb b
bb
a,ba,b
a,ba,b
0
40
80
120
160
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
(mg
/dL
)Creatinine
(Days)
Fig. 3. Serum creatinine and urea con-centrations of diabetic rabbits untreated( ) and treated with either melatonin ( )or N-acetylcysteine (NAC) ( ). Melatoninand NAC were administered as describedin Materials and methods. Values aremean ± S.E.M. for five animals. Dottedlines depict values for control rabbitsequal to 1.3 ± 0.1 mg/dL and30.0 ± 0.2 mg/dL for creatinine and ureaconcentrations respectively. aP < 0.05versus values for untreated diabetic rab-bits, bP < 0.05 versus values for controlanimals.
Table 1. HFR levels and glutathioneredox state in kidney cortex and liver ofcontrol rabbits and diabetic animals bothuntreated and treated with melatonin
Metabolites Organ
Animals
ControlDiabeticuntreated
Diabetic +melatonin
HFR (pmol2,3-DHBA mg d. wt)
Kidney 1.07 ± 0.08 10.97 ± 0.75a 5.68 ± 0.40a, b
Liver 0.76 ± 0.05 5.14 ± 0.36a 2.75 ± 0.17a, b
GSH (nmol/mg d. wt) Kidney 2.64 ± 0.09 2.73 ± 0.09 2.56 ± 0.05Liver 5.56 ± 0.18 4.99 ± 0.21 7.21 ± 0.09a, b
GSSG (nmol/mg d. wt) Kidney 0.051 ± 0.003 0.073 ± 0.004a 0.040 ± 0.002b
Liver 0.058 ± 0.004 0.074 ± 0.003a 0.075 ± 0.005a
GSH/GSSG Kidney 51.8 ± 3.2 37.6 ± 1.5a 54.7 ± 1.4b
Liver 102.0 ± 9.1 66.1 ± 3.4a 98.5 ± 5.5b
2,3-DHBA, 2,3-dihydroxybenzoic acid; HFR, hydroxyl free radical; GSH, reduced gluta-thione; GSSG, oxidized glutathione.All measurements were made following 3 wk of diabetes. Animals intended for HFRdetermination were treated with salicylate 2 hr prior to the experiment. Melatonin wasadministered as described in Materials and methods. Values are mean ± S.E.M. for 3–5animals.aP < 0.05 versus values for control rabbits, bP < 0.05 versus values for untreated diabeticrabbits.
Table 2. Activities of glutathione reduc-tase, glutathione peroxidase and c-glut-amylcysteine synthetase in kidney cortexand liver of control rabbits and diabeticanimals both untreated and treated withmelatonin
Enzyme Organ
Enzyme activity (nmol/min/mg protein)
Controlrabbits
Untreated diabeticrabbits
Diabetic rabbits treatedwith melatonin
Glutathione reductase Kidney 103 ± 12 101 ± 13 146 ± 16a, b
Liver 56 ± 4 64 ± 7 89 ± 10a, b
Glutathione peroxidase Kidney 1573 ± 179 1462 ± 125 1690 ± 87Liver 1793 ± 150 1978 ± 150 5000 ± 395a, b
c-Glutamylcysteinesynthetase
Kidney 987 ± 58 1017 ± 84 990 ± 46Liver 354 ± 22 344 ± 29 434 ± 23a, b
The measurements were made following 3 wk of diabetes. Melatonin was administered asdescribed in Materials and methods. Values are mean ± S.E.M. for three animals.aP < 0.05 versus values for control rabbits, bP < 0.05 versus values for untreated diabeticrabbits.
Winiarska et al.
172
In view of our findings, melatonin seems to be apromising agent for diabetes therapy. Although, in agree-ment with the other reports [38, 45–48], melatonin treat-
ment does not attenuate diabetic hyperglycaemia, iteffectively ameliorates oxidative stress accompanying dia-betes. In this study, treatment of diabetic rabbits with thisantioxidant led to a marked decline in serum, renal and
liver HFR levels (cf. Fig. 1 and Table 1 respectively),confirming that melatonin is a potent HFR scavenger [6,49–51]. Moreover, melatonin administration prevents dia-
betes-evoked disturbances in glutathione homeostasis in thestudied tissues (cf. Fig. 2, Table 1). Similarly, melatonin-induced increase in GSH content has been reported for
kidneys, liver, heart [46], brain [48] and plasma [52] ofstreptozotocin-diabetic rats. The beneficial action of mela-tonin on glutathione homeostasis has also been observedunder other pathological conditions, including chronic
renal failure [53], ischaemia/reperfusion [54, 55] andSchisostoma mansoni infection [56].
In view of the data presented in this paper, we would
suggest that melatonin-evoked changes in glutathionecontent and redox state result from increased activities ofthe enzymes of glutathione metabolism (cf. Table 2). Also,
this phenomenon appears to be tissue dependent. In kidneyof diabetic rabbits melatonin-induced increase in GSH/GSSG ratio is achieved due to the normalization of GSSG
content, which seems to result from the elevated activity ofglutathione reductase. However, in liver melatonin admin-istration leads to the rise in the activities of three enzymesof glutathione metabolism: c-glutamylcysteine synthetase,
glutathione reductase and glutathione peroxidase, affectingboth GSH content and GSH/GSSG ratio. Melatonin-induced enhancement of liver c-glutamylcysteine synthetase
activity might also be responsible for elevated blood GSHcontent observed in melatonin-treated diabetic rabbits. Tosupport our findings, the stimulatory action of melatonin
on c-glutamylcysteine synthetase activity has also beenreported by Urata et al. [57] and Abdel-Wahhab et al. [58].
A nephroprotective action of melatonin deserves special
consideration. As oxidative stress is the main cause ofdiabetic nephropathy [59], administration of antioxidantsappears one of the most reasonable therapeutic approaches.According to our data, melatonin treatment of diabetic
rabbits improves renal function, as concluded from thenormalization of serum creatinine concentration and adecline in serum urea concentration (cf. Fig. 3). Attenu-
ation of diabetic glomerulopathy by melatonin has beenalso reported by Ha et al. [45], who have found thatdiabetic rats treated with this antioxidant exhibited a
lowered rate of renal lipid peroxidation and diminishedurinary protein excretion. Moreover, a decrease in mal-ondialdehyde levels has been observed in kidneys ofstreptozotocin-diabetic rats following several weeks of
melatonin treatment [46, 47]. The indole has also beenpostulated to protect against nephrotoxicity caused byagents other than chronic hyperglycaemia, such as ochra-
toxin [60], adriamycin and constant light exposure [11],cyclosporin A [61], cisplatin [62], gentamicin [63], amikacin[64], mercuric salts [65], ischaemia/reperfusion [66] and
thermal injury [67]. Finally, it should be pointed out thatnegligible toxicity of melatonin [6] additionally indicates the
usefulness of this compound in the therapy of diabetes anddiabetic complications.In contrast to melatonin treatment, NAC administration
to diabetic rabbits fails to attenuate oxidative stress.According to the present findings, NAC does not elevateglutathione content in blood, kidney cortex and liver ofdiabetic animals (cf. Fig. 2 and Table 1 respectively). This
observation seems disappointing, as our previous in vitrostudies have demonstrated that 2 mm NAC significantlyincreases intracellular GSH level in isolated rabbit renal
tubules [5, 15], confirming the high activity of N-deacetylasein kidneys [68]. Moreover, a positive in vivo effect of NACon tissue glutathione content in both humans [69, 70] and
mice [71] has been reported. However, in these investiga-tions NAC was applied at doses several-fold higher thanthose used in the present study. We have chosen the dailydose of 10 mg of NAC per kg body weight to avoid pro-
oxidative [72, 73] and even lethal [74] effects attributed tohigh doses of this compound. It also should be added thatNAC applied at the same dose as in our experiment
ameliorated gentamicin nephrotoxicity in rats [12]. NACaction on glutathione redox state is also controversial. Theresults presented in this investigation (cf. Table 1) seem to
be in agreement with our previous findings that thiscompound does not markedly alter GSH/GSSG ratio inisolated rabbit renal tubules [5, 15]. However, there are also
reports demonstrating NAC-induced elevation of GSH/GSSG ratio both in vitro [75–77] and in vivo [69].A possible nephroprotective action of NAC has been
intensively studied. Under many pathological conditions
NAC treatment resulted in either normalization or at leasta marked decrease in serum creatinine and urea levels [12,78, 79]. Moreover, NAC treatment has been considered
beneficial for patients with renal insufficiency induced byradiocontrast media [80, 81]. Our findings do not confirmNAC nephroprotective effects (cf. Fig. 3), but, as discussed
above, it might result from the relatively low dose of NACadministered to rabbits. However, in the present study, thedose of NAC was 10-fold greater than that of melatonin.
Despite some data indicating a hypoglycaemic action ofNAC [71, 82], we have found no changes in serum glucoselevel of diabetic rabbits treated with this compound. Similarobservations have been made by Pieper et al. [83], who have
studied the effect of NAC administration to streptozotocin-diabetic rats.In summary, data presented in this paper indicate that: (i)
the increased activities of the enzymes of glutathionemetabolism might be responsible for the antioxidativeaction of melatonin, creating this compound much more
effective than NAC in terms of the improvement ofglutathione homeostasis in vivo, and (ii) melatonin admin-istration might be beneficial for the therapy of diabetes anddiabetic complications.
Acknowledgements
The technical assistance of Miss B. Dabrowska is acknow-ledged. We are also very grateful to Dr Z. Bartoszewicz(Medical University of Warsaw) for making available the
electrochemical detector for HFR measurements as well asto Mr K. Wojcik (Brenntag-Polska) for the generous gift of
Melatonin in diabetes treatment
173
silicone oil. This investigation was supported by grants ofthe Ministry of Scientific Research and Information Tech-nology (No. 3 P05A 049 25 and BW 1680/62).
References
1. Bonnefont-Rousselot D, Bastard JP, Jaudon MC et al.
Consequences of the diabetic status on the oxidant/antioxidant
balance. Diabetes Metab 2000; 26:163–176.
2. Brownlee M. Biochemistry and molecular cell biology of
diabetic complications. Nature 2001; 414:813–820.
3. Bryla J, Kiersztan A, Jagielski AK. Promising novel
approaches to diabetes mellitus therapy: pharmacological,
molecular and cellular insights. Eur Citizen’s Qual Life 2003;
1:137–161.
4. Cortizo AM, Bruzzone L, Molinuevo S et al. A possible
role of oxidative stress in the vanadium-induced cytotoxicity in
the MC3T3E1 osteoblast and UMR106 osteosarcoma cell
lines. Toxicology 2000; 147:89–99.
5. Kiersztan A, Winiarska K, Drozak J et al. Differential
effects of vanadium, tungsten and molybdenum on inhibition
of glucose formation in renal tubules and hepatocytes of
control and diabetic rabbits: beneficial action of melatonin and
N-acetylcysteine. Mol Cell Biochem 2004; 261:9–21.
6. Rodriguez C, Mayo JC, Sainz RM et al. Regulation of
antioxidant enzymes: a significant role for melatonin. J Pineal
Res 2004; 36:1–9.
7. Griffith OW. Biologic and pharmacologic regulation of
mammalian glutathione synthesis. Free Radic Biol Med 1999;
27:922–935.
8. Aruoma OI, Halliwell B, Hoey BM et al. The antioxidant
action of N-acetylcysteine: its reaction with hydrogen peroxide,
hydroxyl radical, superoxide, and hypochlorous acid. Free
Radic Biol Med 1989; 6:593–597.
9. Izzotti A, Orlando M, Gasparini L et al. In vitro inhibition
by N-acetylcysteine of oxidative DNA modifications detected
by 32P postlabeling. Free Radic Res 1998; 28:165–178.
10. Kiersztan A, Modzelewska A, Jarzyna R et al. Inhibition
of gluconeogenesis by vanadium and metformin in kidney
cortex tubules isolated from control and diabetic rabbits.
Biochem Pharmacol 2002; 63:1371–1382.
11. Tunez I, Del Carmen Munoz M, Feijoo M et al. Melatonin
effect on renal oxidative stress under constant light exposure.
Cell Biochem Funct 2003; 21:35–40.
12. Mazzon E, Britti D, De Sarro A et al. Effect of N-acetyl-
cysteine on gentamicin-mediated nephropathy in rats. Eur
J Pharmacol 2001; 424:75–83.
13. McCabeDR,Maher TJ, Acworth IN. Improved method for
the estimation of hydroxyl free radical levels in vivo based on
liquid chromatography with electrochemical detection.
J Chromatogr B Biomed Sci Appl 1997; 691:23–32.
14. Dincer Y, Akcay T, Alademir Z et al. Assessment of DNA
base oxidation and glutathione level in patients with type 2
diabetes. Mutat Res 2002; 505:75–81.
15. Winiarska K, Drozak J, Wegrzynowicz M et al. Rela-
tionship between gluconeogenesis and glutathione redox state
in rabbit kidney cortex tubules. Metabolism 2003; 52:739–
746.
16. McLellan LI, Kerr LA, Cronshaw AD et al. Regulation of
mouse glutathione S-transferases by chemoprotectors.
Molecular evidence for the existence of three distinct alpha-
class glutathione S-transferase subunits, Ya1, Ya2, and Ya3, in
mouse liver. Biochem J 1991; 276:461–469.
17. Bergmeyer HU (ed). Methods in Enzymatic Analysis. Verlag
Chemie GmbH: Weinheim, Basel, 1983.
18. Paglia DE, Valentine WN. Studies on the quantitative and
qualitative characterization of erythrocyte glutathione peroxi-
dase. J Lab Clin Med 1967; 70:158–169.
19. Hamilton D, WU JH, Alaoui-Jamali M et al. A novel
missense mutation in the gamma-glutamylcysteine synthetase
catalytic subunit gene causes both decreased enzymatic activity
and glutathione production. Blood 2003; 102:725–730.
20. Kim HG, Hong SM, Kim SJ et al. Age-related changes in the
activity of antioxidant and redox enzymes in rats. Mol Cells
2003; 16:278–284.
21. Cheng FC, Jen JF, Tsai TH. Hydroxyl radical in living sys-
tems and its separation methods. J Chromatogr B Analyt
Technol Biomed Life Sci 2002; 781:481–496.
22. Da Fonseca-Wollheim F, Heinze KG. The influence of
pCO2 on the rate of ammonia formation in blood. Eur J Clin
Chem Clin Biochem 1992; 30:867–869.
23. Michalik M, Biedermann I, Lietz T et al. Recovery of
impaired gluconeogenesis in kidney cortex tubules of gentam-
icin-treated rabbits. Pharmacol Res 1991; 23:259–269.
24. Ridnour LA, Winters RA, Ercal N et al. Measurement of
glutathione, glutathione disulfide, and other thiols in mam-
malian cell and tissue homogenates using high-performance
liquid chromatography separation of N-(1-pyrenyl)maleimide
derivatives. Methods Enzymol 1999; 299:258–267.
25. Layne E. Spectrophotometric and turbidimetric methods for
measuring protein. In: Methods in Enzymology. Colowick SP,
KaplanNO, eds.Academic Press,NewYork, 1957; pp. 447–454.
26. Mori T, Cowley AW, Jr. Renal oxidative stress in medullary
thick ascending limbs produced by elevated NaCl and glucose.
Hypertension 2004; 43:341–346.
27. Verzola D, Bertolotto MB, Villaggio B et al. Oxidative
stress mediates apoptotic changes induced by hyperglycemia in
human tubular kidney cells. J Am Soc Nephrol 2004; 15
(Suppl. 1):S85–S87.
28. Hink U, LI H, Mollnau H et al. Mechanisms underlying
endothelial dysfunction in diabetes mellitus. Circ Res 2001;
88:E14–E22.
29. Ishii N, Patel KP, Lane PH et al. Nitric oxide synthesis and
oxidative stress in the renal cortex of rats with diabetes melli-
tus. J Am Soc Nephrol 2001; 12:1630–1639.
30. Cantero M, Parra T, Conejo JR. Increased hydrogen per-
oxide formation in polymorphonuclear leukocytes of IDDM
patients. Diabetes Care 1998; 21:326–327.
31. Davison GW, George L, Jackson SK et al. Exercise, free
radicals, and lipid peroxidation in type 1 diabetes mellitus.
Free Radic Biol Med 2002; 33:1543–1551.
32. Guzik TJ, Mussa S, Gastaldi D et al. Mechanisms of
increased vascular superoxide production in human diabetes
mellitus: role of NAD(P)H oxidase and endothelial nitric oxide
synthase. Circulation 2002; 105:1656–1662.
33. Martin-Gallan P, Carrascosa A, Gussinye M et al. Bio-
markers of diabetes-associated oxidative stress and antioxidant
status in young diabetic patients with or without subclinical
complications. Free Radic Biol Med 2003; 34:1563–1574.
34. Bhatia S, Shukla R, Venkata Madhu S et al. Antioxidant
status, lipid peroxidation and nitric oxide end products in
patients of type 2 diabetes mellitus with nephropathy. Clin
Biochem 2003; 36:557–562.
35. Erciyas F, Taneli F, Arslan B et al. Glycemic control,
oxidative stress, and lipid profile in children with type 1 dia-
betes mellitus. Arch Med Res 2004; 35:134–140.
Winiarska et al.
174
36. Meral I, Yener Z,Kahraman T et al. Effect of Nigella sativa
on glucose concentration, lipid peroxidation, anti-oxidant
defence system and liver damage in experimentally-induced
diabetic rabbits. J Vet Med A Physiol Pathol Clin Med 2001;
48:593–599.
37. Montilla PL, Vargas JF, Tunez IF et al. Oxidative stress in
diabetic rats induced by streptozotocin: protective effects of
melatonin. J Pineal Res 1998; 25:94–100.
38. Vural H, Sabuncu T, Arslan SO et al. Melatonin inhibits
lipid peroxidation and stimulates the antioxidant status of
diabetic rats. J Pineal Res 2001; 31:193–198.
39. Dominguez C, Ruiz E, Gussinye M et al. Oxidative stress at
onset and in early stages of type 1 diabetes in children and
adolescents. Diabetes Care 1998; 21:1736–1742.
40. Samiec PS, Drews-Botsch C, Flagg EW et al. Glutathione
in human plasma: decline in association with aging, age-related
macular degeneration, and diabetes. Free Radic Biol Med
1998; 24:699–704.
41. Tessier D, Khalil A, Fulop T. Effects of an oral glucose
challenge on free radicals/antioxidants balance in an older
population with type II diabetes. J Gerontol A Biol Sci Med
Sci 1999; 54:M541–M545.
42. Gupta BL, Ansari MA, Singh JN et al. Effect of insulin and
thyroxine on catalase, glutathione-s-transferase, GSH and
GSSG in alloxan diabetic rat red cells. Biochem Int 1992;
27:793–802.
43. Kondo T, Higashiyama Y, Goto S et al. Regulation of
gamma-glutamylcysteine synthetase expression in response to
oxidative stress. Free Radic Res 1999; 31:325–334.
44. Cai J, Sun WM, Lu SC. Hormonal and cell density regulation
of hepatic gamma-glutamylcysteine synthetase gene expres-
sion. Mol Pharmacol 1995; 48:212–218.
45. Ha H, Yu MR, Kim KH. Melatonin and taurine reduce early
glomerulopathy in diabetic rats. Free Radic Biol Med 1999;
26:944–950.
46. Aksoy N, Vural H, Sabuncu T et al. Effects of melatonin on
oxidative-antioxidative status of tissues in streptozotocin-in-
duced diabetic rats. Cell Biochem Funct 2003; 21:121–125.
47. CamM, Yavuz O,GuvenA et al. Protective effects of chronic
melatonin treatment against renal injury in streptozotocin-in-
duced diabetic rats. J Pineal Res 2003; 35:212–220.
48. Baydas G, Reiter RJ, Yasar A et al. Melatonin reduces glial
reactivity in the hippocampus, cortex, and cerebellum of
streptozotocin-induced diabetic rats. Free Radic Biol Med
2003; 35:797–804.
49. Mahal HS, Sharma HS, Mukherjee T. Antioxidant prop-
erties of melatonin: a pulse radiolysis study. Free Radic Biol
Med 1999; 26:557–565.
50. Tan DX, Reiter RJ, Manchester LC et al. Chemical and
physical properties and potential mechanisms: melatonin as a
broad spectrum antioxidant and free radical scavenger. Curr
Top Med Chem 2002; 2:181–197.
51. Winiarska K, Drozak J, Wegrzynowicz M et al. Diabetes-
induced changes in glucose synthesis, intracellular glutathione
status and hydroxyl free radicals generation in rabbit kidney
cortex tubules. Mol Cell Biochem 2004; 261:91–98.
52. Gorgun FM, Kokoglu E, Gumustas MK et al. Effects of
melatonin on plasma S-nitrosoglutathione and glutathione in
streptozotocin-treated rats. J Toxicol Environ Health A 2004;
67:979–986.
53. Sener G, Paskaloglu K, Toklu H et al. Melatonin ameli-
orates chronic renal failure-induced oxidative organ damage in
rats. J Pineal Res 2004; 36:232–241.
54. Sener G, Sehirli AO, Keyer-Uysal M et al. The protective
effect of melatonin on renal ischemia-reperfusion injury in the
rat. J Pineal Res 2002; 32:120–126.
55. Ozacmak VH, Sayan H, Arslan SO et al. Protective effect of
melatonin on contractile activity and oxidative injury induced
by ischemia and reperfusion of rat ileum. Life Sci 2005;
76:1575–1588.
56. El-SokkaryGH, OmarHM,HassaneinAF et al. Melatonin
reduces oxidative damage and increases survival of mice
infected with Schistosoma mansoni. Free Radic Biol Med 2002;
32:319–332.
57. Urata Y,Honma S,Goto S et al. Melatonin induces gamma-
glutamylcysteine synthetase mediated by activator protein-1 in
human vascular endothelial cells. Free Radic Biol Med 1999;
27:838–847.
58. Abdel-Wahhab MA, Abdel-Galil MM, El-Lithey M.
Melatonin counteracts oxidative stress in rats fed an ochra-
toxin A contaminated diet. J Pineal Res 2005; 38:130–135.
59. Lehmann R, Schleicher ED. Molecular mechanism of dia-
betic nephropathy. Clin Chim Acta 2000; 297:135–144.
60. Ozcelik N, Soyoz M, Kilinc I. Effects of ochratoxin a on
oxidative damage in rat kidney: protective role of melatonin.
J Appl Toxicol 2004; 24:211–215.
61. Longoni B, Migliori M, Ferretti A et al. Melatonin pre-
vents cyclosporine-induced nephrotoxicity in isolated and
perfused rat kidney. Free Radic Res 2002; 36:357–363.
62. Parlakpinar H, Sahna E, Ozer MK et al. Physiological and
pharmacological concentrations of melatonin protect against
cisplatin-induced acute renal injury. J Pineal Res 2002; 33:161–
166.
63. Ozbek E, Turkoz Y, Sahna E et al. Melatonin administra-
tion prevents the nephrotoxicity induced by gentamicin. BJU
Int 2000; 85:742–746.
64. ParlakpinarH, OzerMK, Sahna E et al. Amikacin-induced
acute renal injury in rats: protective role of melatonin. J Pineal
Res 2003; 35:85–90.
65. Sener G, Sehirli AO, Ayanoglu-Dulger G. Melatonin
protects against mercury(II)-induced oxidative tissue damage
in rats. Pharmacol Toxicol 2003; 93:290–296.
66. Rodriguez-Reynoso S, Leal C, Portilla-De Buen E et al.
Melatonin ameliorates renal ischemia/reperfusion injury.
J Surg Res 2004; 116:242–247.
67. SenerG, Sehirli AO, Satiroglu H et al. Melatonin prevents
oxidative kidney damage in a rat model of thermal injury. Life
Sci 2002; 70:2977–2985.
68. Yamauchi A, Ueda N, Hanafusa S et al. Tissue distribution
of and species differences in deacetylation of N-acetyl-L-cys-
teine and immunohistochemical localization of acylase I in the
primate kidney. J Pharm Pharmacol 2002; 54:205–212.
69. De Mattia G, Bravi MC, Laurenti O et al. Reduction of
oxidative stress by oral N-acetyl-L-cysteine treatment decrea-
ses plasma soluble vascular cell adhesion molecule-1 concen-
trations in non-obese, non-dyslipidaemic, normotensive,
patients with non-insulin-dependent diabetes. Diabetologia
1998; 41:1392–1396.
70. Roes EM, Raijmakers MT, Peters WH et al. Effects of oral
N-acetylcysteine on plasma homocysteine and whole blood
glutathione levels in healthy, non-pregnant women. Clin Chem
Lab Med 2002; 40:496–498.
71. Ho E, Chen G, Bray TM. Supplementation of N-acetylcy-
steine inhibits NFkappaB activation and protects against all-
oxan-induced diabetes in CD-1 mice. FASEB J 1999; 13:1845–
1854.
Melatonin in diabetes treatment
175
72. Oikawa S, Yamada K, Yamashita N et al. N-acetylcysteine,
a cancer chemopreventive agent, causes oxidative damage to
cellular and isolated DNA. Carcinogenesis 1999; 20:1485–
1490.
73. Sagrista ML, Garcia AE, Africa De Madariaga M et al.
Antioxidant and pro-oxidant effect of the thiolic compounds
N-acetyl-L-cysteine and glutathione against free radical-in-
duced lipid peroxidation. Free Radic Res 2002; 36:329–340.
74. Sprong RC, Winkelhuyzen-Janssen AM, Aarsman CJ
et al. Low-dose N-acetylcysteine protects rats against endo-
toxin-mediated oxidative stress, but high-dose increases mor-
tality. Am J Respir Crit Care Med 1998; 157:1283–1293.
75. Dobashi K, Aihara M, Araki T et al. Regulation of LPS
induced IL-12 production by IFN-gamma and IL-4 through
intracellular glutathione status in human alveolar macroph-
ages. Clin Exp Immunol 2001; 124:290–296.
76. Sanz-Alfayate G, Obeso A, Agapito MT et al. Reduced
oxidized glutathione ratios and oxygen sensing in calf and
rabbit carotid body chemoreceptor cells. J Physiol 2001;
537:209–220.
77. Victor VM, Rocha M, de la Fuente M. N-acetylcysteine
protects mice from lethal endotoxemia by regulating the redox
state of immune cells. Free Radic Res 2003; 37:919–929.
78. Sehirli AO, Sener G, Satiroglu H et al. Protective effect of
N-acetylcysteine on renal ischemia/reperfusion injury in the
rat. J Nephrol 2003; 16:75–80.
79. Hsu BG, Yang FL, Lee RP. N-acetylcysteine ameliorates
lipopolysaccharide-induced organ damage in conscious rats.
J Biomed Sci 2004; 11:152–162.
80. Pannu N, Manns B, Lee H et al. Systematic review of the
impact of N-acetylcysteine on contrast nephropathy. Kidney
Int 2004; 65:1366–1374.
81. Kshirsagar AV, Poole C, Mottl A et al. N-acetylcysteine
for the prevention of radiocontrast induced nephropathy: a
meta-analysis of prospective controlled trials. J Am Soc
Nephrol 2004; 15:761–769.
82. Kaneto H, Kajimoto Y, Miyagawa J et al. Beneficial effects
of antioxidants in diabetes: possible protection of pancreatic
beta-cells against glucose toxicity. Diabetes 1999; 48:2398–
2406.
83. Pieper GM, Siebeneich W. Oral administration of the anti-
oxidant, N-acetylcysteine, abrogates diabetes-induced endot-
helial dysfunction. J Cardiovasc Pharmacol 1998; 32:101–105.
Winiarska et al.
176
