J. Physio!. Biochem., 58 (2), 109-114,2002

Effect of melatonin on hyperlipidemic nephropathy under constant light exposure I. Tunez, M. c. Munoz, M. Feijoo-Lopez, E. Valdelvira, I. Bujalance-Arenas and P. Montilla Department of Biochemistry and Molecular Biology, School of Medicine, University of Cordoba, 14004Cordoba, Spain (Receivedon Feburary 26, 2002)

1. TONEZ, M. c. MUNOZ, M. FEI]OO-L6PEZ, E. VALDELVlRA, 1. BU]ALANCE-ARENAS and P. MONTILLA. Effect of melatonin on hyperlipidemic nephropathy under constant light exposure. ]. Physio!. Biochem., 58 (2), 109-114,2002. Studies have shown anti-hyperlipidemic actions of melatonin, with pharmacological doses inducing changes in cholesterol levels. This study was designed to evaluate the effect of melatonin on adriamycin-induced (25mg/kg b.w., i.p.) hyperlipidemia under constant light exposure. Melatonin was injected i.p. (toOO ug/kg b.w.lday). Triglycerides, total cholesterol, high-density lipoprotein cholesterol, light-density lipoprotein cholesterol, non-proteic nitrogen compounds (urea and creatinine levels), total protein in serum, proteins eliminated in the urine and melatonin levels in serum and kidney were determined. Results show a decrease in melatonin levels induced by both adriamycin and constant light. Likewise, adriamycin induced significant increases in triglycerides, total cholesterol and light-density lipoprotein cholesterol, and lowered high-density lipoprotein cholesterol levels. Constant light exposure also prompted an increase in LDL-c levels and a decrease in HDL-c values, and intensified the effects of adriamycin on these two lipoproteins. All changes induced by adriamycin and constant light were reverted toward normality by melatonin administration. Key words: Melatonin, Hyperlipidemia, Lipid profile, Constant light.

Melatonin, the main product of the vertebrate pineal gland, is probably best know for its effects on seasonal reproductive physiology in photoperiodic mammals (14) and in circadian rhythmicity (1). Correspondence to P. Montilla(Tel.: 34 957218 268; Fax: 34 957 218 229; e-mail: [email protected]; [email protected]).

Indirect evidence also suggests that melatonin may augment endogenous cholesterol clearance mechanisms and lower total cholesterol. This is accompanied by the lowering of the cholesterol fraction associated with low-density lipoproteins (LDL-c) and by the enhancing of the high-density lipoprotein cholesterol (HDL-c) (6, 8, 12, 15).

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Adriarnycin, commonly used as an anti-tumoral antibiotic in humans, has been demonstrated to be an excellent model to reproduce syndromes of hyperlipidemic nephropathy accompanied by oxidative stress in rats and other rodents that are quite similar to human focal-segmental glomerulosclerosis (2-4, 7, 13, 17). Many studies support the beneficial and protective effects of antioxidants in these pathologies (9, 16). Melatonin, in accord with research carried out by our group, significantly reduces the intensity of oxidative stress, hyperlipidemic and signs of renal disruption induced by adriamycin in rat (10, 11). Considering these data in relation with melatonin and hyperlipidemia, our group designed the present study with the aim to evaluate the effects of melatonin and constant light exposure on lipid profile (triglycerides, total cholesterol, LDL-c and HDL-c) in adriamycin-induced hyperlipidemia, in order to be able establish a direct relation between melatonin levels and adriamycin-induced hyperlipidemia. Materials and Methods

Animals and experimental design.Three month old male Wistar rats, weighing 300 g, were purchased from Charles River (Crediffa S.A., Barcelona, Spain). During the study, the animals were subjected to controlled conditions of temperature (20-23 "C), feeding (Purina, Barcelona, Spain), water ad libitum and illumination (14 h light/IO h darkness), and some animals were kept under constant light as described below. Six animals were included in each of the following nine groups: 1) control, 2) vehicle, 3) melatonin (MEL), 4) constant light (CL), 5) constant light + melatonin, 6) J. Physio!. Biochem., 58 (2), 2002

adriamycin (AD), 7) adriamycin + melatonin, 8) adriamycin + constant light, and 9) adriamycin + constant light + melatorun. Crystalline melatonin solution 1000 pg/kg/b.w./i.p. was prepared daily, using ethanol at 5% in NaCI 0.9% saline, whereas adriamycin was administered i.p. at 25mg/kg/b.w. To measure constant light effects on lipid profile some animals were exposed to constant light for seven days. On the 7th day, under ether anaesthesia, the animals were sacrificed and trunk blood and kidneys were collected. Kidneys were dissected and the parts were frozen (-80° C) until the parameters measurement was performed as previously described (5). The renal blood was removed from the kidney before homogenrzing.

Biochemical determinations.- Melatonin levels were determined in both serum and renal tissue by rat-specific radioimmunoassay (Peninsula S.A., USA). The serum levels of the urea, creatinine and lipid profile were determined enzymatically using kits and procedures obtained from Boehringer Mannheim (Barcelona, Spain). Proteins in urine were measured with the biuret reagent (BioMerieux S.A., Spain). Melatonin (N-acetyl-S-methoxytryptamine) was supplied by Sigma Chemical Co. (St. Louis, USA); and absolute ethanol from Merck (Barmstadt, Germany). Statistical analysis.- The values express the means ± S.E.M., whose significance was estimated according to the ANOVA and Student's t-test, means were considered to differ significantly if p< 0.05.

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Results Baseline and constant light exposure.Exposure of rats with or without adriamycin-induced hyperlipidemia to constant light exposure led to a significant increase in LDL-c levels (Table I), and prompted a significant fall in HDL-c concentrations (Table I). Exposure to constant light also had a marked effect on the melatonin levels (Fig. 1), while constant light exposure prompted no changes in serum urea, creatinine, and total protein nor in urinary protein excretion (Table II). Effect of melatonin administration.Administration of exogenous melatonin in the control and adriamycin groups caused a significant drop in LDL-c and an increase in HDL-c (Table I). Treatment with melatonin, in addition to reducing and increasing the LDL-c and HDL-c, respectively, prompted a significant decrease in triglycerides and total cholesterol in the adriamycin groups (Table I).

This considerable improvement in the hyperlipidemia marker values following administration of melatonin in animals with adriamycin-induced nephropathy and exposed to constant light was associated with an increase in serum and renal tissue endogenous melatonin levels (Fig. 1). Also, melatonin reverted toward normality the changes induced by adriamycin in urea, creatinine, total protein in serum, and excretion of urinary proteins (Table II).

Discussion These results demonstrated an increase in LDL-c and a decrease in HDL-c in experimental nephropathy induced by adriamycin under constant light exposure. This imbalance was evident not only in adriamycin plus constant light group, but also in healthy animals exposed to constant light. Secretion of melatonin by the pineal gland displays a circadian rhythm, with peak levels occurring at night (between

Table I. Effect of melatonin and constant light on the changes of lipid profile (triglycerides, total choles-

terol, HDL-c and LDL-c) induced by adriamycin. Values are expressed as mean ± S.E.M. (N=6).

Triglycerides (mg/dl) Control Vehicle Melatonin Constant light + Melatonin Adriamycin + Melatonin + Constant light + Constant light + Melatonin

120 116 124 118 108 275 153 278 163

±

4.72

± 3.82 ± 4.50

3.25 3.27 ± 3.44 a ± 5.14 c ± 5.67 ± 5.86 e ± ±

Total cholesterol (mg/dl)

89.7 ± 2.91 88.0 ± 2.42 87.0 ± 3.42 81.0 ± 2.30 81.1 ± 2.25 279.0 ± 11.4 a 89.7±9.10c 389.1 ± 9.9 86.0 ± 6.90 e

HDL-c (mg/dl)

55.0 57.6 75.0 49.3 73.4 30.0 49.7 26.4 58.3

± ±

± ± ±

±

± ± ±

1.81 4.38 2.64 a 2.72 a 5.34 b 0.57 a 1.47 c 0.62 d 1.82 e

LDL-c (mg/ dl)

45.0 46.3 10.0 53.2 8.0 84.0 15.4 98.1 11.8

± 1.46

± 2.34 ± 0.35 a ± 1.53 a ± 0.79 b ± 0.57 a ± 0.90 c ± 2.47 d ± 1.08 e

a p < 0.001 with respect control group; b p < 0.001 with respect constant light group; C p < 0.001 with respect adriamycin group; d p < 0.01 with respect adriamycin group; e p < 0.001 with respect adriamycin + constant light group.

J. Physio!. Biochem., 58 (2),2002

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Fig. 1. Melatonin levels in serum and kidney in all the groups studied. < 0.001 us. control group (values in vehicle group are equivalent to to that of the control group). ... p < 0.001 us. constant light (CL.) group. +++ p < 0.001 us. adriamycin (A) group. 000 p < 0.001 us. adriamycin + constant light group. Values are expressed as mean ± S.E.M.; n = 6 rats in each group. M: melatonin.

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Table II. Effect of melatonin and constant fight on nonproteic nitrogen compounds (urea and creatinine),

total protein and urinary protein excretion in rats. Values are expressed as mean ± S.E.M. (n=6).

Urea (mg/dl) Control Vehicle Melatonin Constant light + Melatonin Adriamycin + Melatonin + Constant light + Constant light + Melatonin

35.7 ± 36.1 ± 36.0 ± 37.1 ± 36.9 ± 70.5 ± 28.2 ± 71.4 ± 39.6 ±

0.87 1.05 1.90 0.93 1.25 1.37 a 0.95 c 1.36 1.23 d

Creatinine (mg/dl)

0.8 ± 0.01 0.9 ± 0.03 0.6 ± 0.02 0.7 ± 0.05 0.8 ± 0.03 4.1 ± 0.14 a 1.4 ± 0.08 c 3.8 ± 1.02 1.6±0.10d

Total proteins Urinary protein (mg/dl) (mg/ dl)

6.6 6.9 6.8 6.9 6.6 4.5 6.8 4.1 6.3

a p < 0.001 with respect control group; C p < 0.001 with respect adriamycin group; amycin + constant light group.

J. Physiol. Biochem., 58 (2), 2002

d

± 0.14 ± 0.43 ± 0.29 ± 0.18 ± 0.21 ± 0.16 a ± 0.27 c ±0.44 ± 0.27 d

6.7 ± 0.01 6.5 ± 0.03 6.7 ± 0.01 6.5 ± 0.02 6.5 ± 0.03 29.1 ± 0.21 a 9.08 ± 0.07 c 28.2 ± 0.05 8.9 ± 0.08 d

p < 0.001 with respect adri-

MELATONIN AND HYPERLIPIDEMIA

02:00 of 04:00). Both synthesis and release of melatonin are stimulated by darkness and inhibited by light. Inhibition of melatonin synthesis by constant light exposure, evident here in a drop in serum and renal tissue levels, may also account for the worsening of the already-impaired lipid balance found in nephropathy induced by adriamycin. In the present study, constant light exposure, an experimental condition that induced a drop in melatonin levels, prompted a fall in HDLc levels and enhanced in LDL-c levels. There was also an increase in triglycerides and ~otal cholesterol induced by adriamyclll. In order to limit lipid profile imbalance in nephropathy induced by adriamycin aggravated by the effect of light, animals received exogenous melatonin. Although endogenous melatonin was inhibited by constant light exposure, administration of melatonin at pharmacological levels prompted significantly elevated serum and renal tissue melatonin levels, decreased LDL-c levels and led to a substantial recovery of HDL-c, establishing a direct relationship between the levels of peripheral melatonin and lipid profile. In previous studies, our group found melatonin to be an effective anti-hyperlipidemic in adriamycin-induced nephropathy. These studies showed that melatonin lowered triglycerides, phospholipids and total cholesterol levels and increased HDL-c levels in experimental hyperlipidemia induced by adriamycin (10, 11), findings confirmed by the present study. We have also observed that melatonin is more effective in hyperlipidemia than vitamin E (10). In the present study we confirmed the melatonin effect on the levels of LDL-c, HDL-c and total cholesterol, data that indicate an action both endogenous and exogenous melatonin on J. Physio!. Biochem., 58 (2),2002

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the cholesterol clearance mechanisms. Similar findings were also reported for other experimental models, which revealed a decline in LDL-c and total cholesterol, and enhanced in HDL-c in animals treated with melatonin (6, 8, 12, 15). However, the melatonin levels and constant light exposure recorded here were not a factor in the other study. In conclusion, the results obtained suggest an increase in LDL-c and a decrease in HDL-c in animals with or without experimentally-induced nephropathy exposed to constant light; the increase is reverted by administration of exogenous melatonin. Moreover, with respect to the biochemical signs indicative of nephropathy, melatonin prompted significant decreases in serum urea, creatinine, total proteins and proteinuria induced by adriamycin. Particularly important is the decrease in proteinuria, a primary biochemical sign in the evaluation of the intensity and course of nephropathy induced by adriamycin. Finally, these data establish a clear relationship between melatonin levels and adriamycin-induced hyperlipidemia. For these reasons we are continuing to investigate the effects of melatonin on hyperlipidemia. 1. TONEZ, M. c. MUNOZ, M. FEIJOOL6PEZ, E. VALDELVIRA, 1. BUJALANCE-ARENAS Y P. MONTILLA. NeJropatia biperlipidemica tras iluminaci6n canstante: ejecta de la melatanina. J. Physiol. Biochem., 58 (2), 109-114, 2002. Se ha descrito que la melatonina presenta accion antihiperlipidernica y que, a dosis farmacologica, modifica los niveles de colesterol. El objetivo del presente trabajo consiste en evaluar el efecto de la melatonina sobre la hiperlipidemia inducida por adriamicina (25 mg/kg peso corporal, i.p.), bajo iluminacion constante. La melatonina se inyecta por via intraperitoneal, 100 mg/kg peso corporal. Se determinan los niveles sericos de trigliceridos,

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colesterol total, HDL-colesterol, LDL-colesterol, urea y creatinina, las proteinas eliminadas en orina y los niveles de melatonina en suero y en rifion, Los resultados muestran que por efecto conjunto de la adriamicina y la iluminaci6n constante se observa disminuci6n de la melatonina. La adriamicina incrementa los niveles de trigliceridos, colesterol total y LDLcolesterol y disminuye los de HDL-colesterol. La iluminaci6n constante tambien modifica en el mismo sentido los niveles de HDL y LDL e intensifica los efectos de la adriamicina sobre ambas lipoproteinas. Todos los cambios inducidos por adriamicina y por iluminaci6n constante revierten hacia la normalidad por administraci6n de melatonina. Palabras clave: Melatonina, Hiperlipidernia, Perfil lipidico, Iluminacion constante.

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