Send Orders for Reprints to
[email protected] 126
Endocrine, Metabolic & Immune Disorders - Drug Targets, 2014, 14, 126-133
Assessment of a Synthetic Steroid and Flutamide on Dopamine, GSH and H2O2 Levels in Rat Brain in Presence of Fructose David C. Guzmán1, Eugene Bratoeff 2, Aylin S. Ortíz2, Ernestina H. García3, Norma O. Brizuela1, Gerardo B. Mejía1, Hugo J. Olguín3,*, Daniel S. del Angel1 and Israel M. Cruz1 1
Laboratorio de Neuroquímica, Instituto Nacional de Pediatría (INP), México; 2Laboratorio de Química Farmacéutica, Facultad de Química, Universidad Nacional Autónoma de México (UNAM), México; 3Laboratorio de Farmacología, INP. Facultad de Medicina UNAM, México Abstract: Flutamide is a drug used in the treatment of androgen-dependent disorders. However, this treatment is usually accompanied by some adverse side effects. The aim of this work was to analyse the effect of flutamide and to compare this effect with that of a synthetic steroid - 3β-propionyloxy-5-androsten-17-one (PPA) - on levels of dopamine and some oxidative stress markers. For this, thirty-six male young Wistar rats (65g) were recruited and divided into 6 groups. The groups were then treated as follows: Group 1 (control), dimethyl sulfoxide (DMSO); group 2, flutamide (4mg/kg); group 3, PPA; group 4, DMSO + fructose; group 5, flutamide + fructose; and group 6, PPA + fructose. The treatments were administered intraperitoneally at a daily dose of 4 mg/kg for 10 days. In the last day of treatment, blood samples were obtained and used to assess the levels of glucose and cholesterol. The animals were then sacrificed and their prostate gland and brains were obtained for measurement of 5α-reductase, glutathione (GSH), calcium, H2O2, and dopamine in cortex, hemispheres, and medulla/oblongata, using previously validated methods. Results: Dopamine levels decreased while GSH increased significantly in cortex and hemispheres of animals that received PPA plus fructose. Also in the same group, GSH decreased in cerebellum/medulla oblongata when compared with control group. Peroxidation decreased significantly in all tissues of the groups, while ATPase activity witnessed a significant decrease in cortex and an increase in hemispheres of animal groups treated with flutamide and PPA both in combination with fructose. Conclusion: The steroid, 3β-propionyloxy-5-androsten-17-one, may in part act as a neuroprotector mediated by the increase of GSH and decrease of H2O2. Besides, imbalance in steroid homeostasis may alter the metabolism of dopamine.
Keywords: Brain, oxidative stress, peroxidation, prostate, rat, synthetic steroid. INTRODUCTION The neural androgen receptor as a therapeutic target for myelin recovery may provide the preclinical rationale for a therapeutic use of androgens in males with neurological disorders [1]. The incorporation of new drugs to treat androgen-dependent situations [2] includes drugs which have been successful in prostate cancer remission, which apart from inhibiting 5α-reductase enzyme necessary for transforming testosterone to dihydrotestosterone (DHT), are intended to inhibit 17α-hydroxylase and C17, 20 lyase enzymes [3]. Although, the activity of aromatase in the brain declines after exposures to flutamide [4], there is an evidence that testosterone and DHT link to common receptor, and that the effect of DHT is not inhibited by testosterone. Besides, DHT increases androgenic effect because it has major affinity for androgenic receptor (AR) [5]. Recently, AR has
*Address correspondence to this author at the Laboratorio de Farmacología, Instituto Nacional de Pediatría, Av Iman Nun 1, 3 er piso, Col Cuicuilco, CP 04530, Mexico City, Mexico; Tel/Fax: 5255 1084 3883; E-mail
[email protected] 2212-3873/14 $58.00+.00
been involved in the neurogenesis of brain hippocampus pathway [6]. The conversion of testosterone to DHT enhances androgenic effect. Such increase may produce a poor androgenic effect in tissues where there is a low amount of testosterone, although that could be important for DHTdependent tissues [7]. Indeed, imbalances in steroid homeostasis may impair the signaling of dopamine (DA) and other neurotransmitters. Besides, some hormone-related satiety is increased with fructose ingestion [8]. The pathogenesis of Tourette syndrome (TS) is thought to reflect deregulations in the signaling of dopamine and other neurotransmitters leading to excitation/inhibition imbalances in cortico-striato-thalamocortical circuits [9]. Any strategy aimed to obtain compounds that could diminish DHT levels should offer positive therapeutic response with minimal adverse effects [10]. Previous studies carried out in our laboratory showed that opioid receptors alter endogenous nitric oxide (NO) levels in some brain tissues [11], and that there is steroid participation in the regulation of this biomarker. NO is a neuromodulator, © 2014 Bentham Science Publishers
Effect of New Steroid on Brain and Prostate
Endocrine, Metabolic & Immune Disorders - Drug Targets, 2014, Vol. 14, No. 2
however, high levels if this may lead to cell damage produced by oxidative stress or by forming nitrosoglutathione (NOGSH) inside the cell [12]. It is known that free radicals can produce damage to cell components [13], principally to plasma membrane lipids [14]; and that central nervous system (CNS) is very susceptible and dependent on the amount of antioxidants, particularly during development of the organism, when brain metabolism and growth rates are high [15]. Fructose promotes changes in the brain reward system, which leads to excessive consumption and also promotes reactive oxygen species formation [16]. Plasma membrane phospholipids in brain are in contact with structural proteins that are inserted in the lipid bilayer [17], in which Na+, K+ ATPase acts to ensure ionic interchange by stimulating Na+ and K+ flows [18]. The inhibition of Na+, K + ATPase enzyme activity induces excitatory amino acid release within CNS [19]. Based on this background, the purpose of this study was to compare the effect of flutamide and a synthetic steroid (3β-propionyloxy-5-androsten-17-one) on levels of dopamine and some oxidative stress markers in brain of young rats and prostate in presence of fructose. MATERIAL AND METHODS Thirty-six male young Wistar rats (65g) were divided into 6 groups and treated as follows: Group 1 (control), dimethyl sulfoxide (DMSO); group 2, flutamide (4mg/kg); group 3, 3β-propionyloxy-5-androsten-17-one (PPA) (Fig. 1A); group 4, DMSO + fructose; group 5, flutamide + fructose; and group 6, PPA + fructose. The fructose at 10% was diluted in water and administered at liberty. All drugs were intraperitoneally administered every 24 hours, for ten days. On the last day of treatment, samples of blood were taken and immediately used to measure glucose and cholesterol levels. Then, the animals were sacrificed and their prostate glands and brains were extracted and stored in saline solution at 4°C. Each section of the brain was sagitally dissected and the left-side slice was homogenised in 5 volumes of 0.05M TRIS-HCl, pH 7.4 to determine 5 αreductase, ATPase and H2O2 while the right-side slice was homogenised in 5 volumes of 0.1M perchloric acid (HClO4) and used to evaluate reduced glutathione (GSH), dopamine, and calcium concentrations. Extraction of Prostate Gland The sacrificed rats were ventrally positioned and an incision through the infra-supra umbilical plane was made to
expose the urinary bladder. Then, an incision was made in the base of the prostate gland and the organ was extracted. This was weighed and homogenised using ultrasonic sonicator (Vibra Cell, Sonic and Materials Inc. USA), with three 5-second lapses at 60 Htz, and stored at –20°C until analysed [7]. Animal management and care were in accordance with international guidelines for animal care and Mexican Guidelines ZOO-062. Technique to Measure Blood Glucose and Cholesterol The measurement of glucose and cholesterol was carried out at the end of treatment. Two blood samples each 20µl drawn from the tail-end without anticoagulant were placed on Accu-Chek (Roche Mannheim, Germany) equipment reactive paper to measure the concentrations of glucose and cholesterol and reported in mg/dl. Measurement of Dopamine The levels of dopamine were measured in the supernatant of tissue homogenized in HClO4 after centrifugation at 9,000 rpm for 10min in a microcentrifuge (Hettich Zentrifugen, model Mikro 12-42, Germany), with a version of the technique reported by Calderon et al. [20]. An aliquot of the HClO4 supernatant, and 1.9 ml of buffer (0.003M octylsulphate, 0.035M KH2PO4, 0.03M citric acid, 0.001M ascorbic acid), were placed in a test tube. The mixture was incubated for 5 min at room temperature in total darkness, and subsequently the samples were read in a spectrofluorometer (Perkin Elmer LS 55, England) with 282 nm excitation and 315 nm emission lengths. The FL Win Lab version 4.00.02 software was used. Values were inferred in a previous standardized curve and reported as nMoles/g of wet tissue. Measurement of Glutathione (GSH) GSH levels were measured from the supernatant of the homogenised tissue, which was obtained after centrifuging at 9000 rpm during 5 minutes (Mikro 12-42, Germany centrifuge) based on a modified method of Hissin and Hilf [21]. 1.8 mL phosphate buffer pH 8.0 with EDTA 0.2%, 20 µL taken from the supernatant and 100 mL of orthophthaldehyde at 1mg/mL in methanol were mixed in a test tube, incubated for 15 min at room temperature in absolute darkness and were spectrophotometrically read with PERKIN ELMER LS 55, with excitation and emission wavelengths of 350 and 420, respectively. FL Win Lab version 4.00.02 software was used. Values were inferred from a previously standardized curve and expressed as nM/g.
O
0 H O
H O
127
H H
H HO
Fig. (1). A. 3β-propionyloxy-5-androsten-17-one (PPA) B. Dehydroepiandrosterone (DHEA).
H
128 Endocrine, Metabolic & Immune Disorders - Drug Targets, 2014, Vol. 14, No. 2
Table 1.
Levels of cholesterol and glucose in blood of rats treated with flutamide and 3β-propionyloxy-5androsten-17-one (PPA) in presence of fructose (F). Mean ± SD.
Groups
Cholesterol (mg/dL)
Glucose (mg/dL)
Control (DMSO)
158.6 ± 6.4
140 ± 17.7
Flutamide
155.6 ± 6.8
145.6 ± 19.4
PPA
156.3 ± 4.7
128.6 ± 26.6
F + DMSO
158.8 ± 7.0
141.3 ± 11.3
F + Flutamide
156.5 ± 5.7
138.5 ± 6.12
F + PPA
156.3 ± 3.4
133.17 ± 7.86
DMSO: Dimethyl sulfoxide
Measurement of Calcium The procedure to measure calcium was performed using supernatant liquid from the brain homogenate for each brain tissue of all animal groups, using Ca-Color Arsenazo III AA direct colorimetric method kit (Wiener Lab Rosario, Argentina). The concentration was obtained using an internal standard and was reported as mg/g wet tissue. Measurement of Steroid 5α-Reductase Activity The enzymatic activity was determined using 1mg of protein from the supernatant of rat brain regions and incubated at 37°C obtaining a final concentrations of NADPH (0.8mM) and testosterone (4mM) in a shaking water bath. The decrease of absorbance was monitored at 340nm (NADPH) for 30minutes in a Helios-α, UNICAM spectrophotometer [22]. Measurement of Total ATPase The activity of ATPase was assayed according to the method proposed by Calderón et al. [22]. 1 mg (10%) w/v of homogenised brain tissue in tris-HCl 0.05M pH 7.4 was incubated for 15 min in a solution containing 3mM MgCl2 , Table 2.
Guzmán et al.
7mM KCl, and 100mM NaCl. To this was added 0.5, 1, 2, 3, and 4 mM tris-ATP and incubated for another 30 min at 37oC in a shaking water bath (Dubnoff Labconco). 100 µL 10% trichloroacetic acid w/v was used to stop the reaction and samples were centrifuged at 100 g for 5 minutes at 4oC. Inorganic phosphate (Pi) was measured in duplicates using one supernatant aliquot as reported by Fiske and Subarrow [23]. The absorbance of a supernatant sample was read at 660 nm in a Helios-α, UNICAM spectrophotometer and expressed as mM Pi/g wet tissue per minute. Measurement of H2O2 The determination of H2O2 was made using the modified technique [24]. Each brain region (cortex, hemispheres, cerebellum/medulla oblongata) was homogenized in 3 ml of tris-HCl 0.05M pH 7.4 buffer. From the diluted homogenates, 100µl was taken and added to 1ml of potassium dichromate solution (K2Cr2O7). The mixture was heat to boiling point for 15min. (Thermomix 1420). The samples were later placed in an ice bath for 5 min and then centrifuged at 3,000g for 5min. (Sorvall RC-5B Dupont). The absorbances of the floating were read in triplicate at 570nm in a spectrophotometer (Heλios-α, UNICAM). The concentration of H2O2 was expressed in µMoles. Statistical Analysis Analysis of variances (ANOVA) and the Kruskal-Wallis test were used with their corresponding contrasts, and previous variance homogeneity comparison. Values of p
