Supplementary information
Interaction of curcumin with manganese compromises metal and neurotransmitter homeostasis in the hippocampus of young mice
Ariana Ern Schmitza, Paulo Alexandre de Oliveirab, Luiz F. de Souzaa, Danilo Grünigc, Samara Danielskia, Danúbia Bonfanti dos Santosa, Eduardo Alves de Almeidac, Rui Daniel Schröder Predigerb, Andrew Fisherd, Marcelo Farinaa, Alcir Luiz Dafrea,*
aDepartment of Biochemistry, Federal University of Santa Catarina, Florianópolis, SC, Brazil
bDepartment of Pharmacology, Federal University of Santa Catarina, Florianópolis, SC, Brazil
cDepartment of Chemistry and Environmental Sciences, University Paulista Júlio de Mesquita Filho, SP, Brazil
dSchool of Geography, Earth and Environmental Sciences, University of Plymouth, PL4 8AA Plymouth, United Kingdom
Corresponding author. Address: Federal University of Santa Catarina, Department of Biochemistry, Biological Sciences Centre, 88040-900, Florianópolis, SC, Brazil. fax: +55 48 3721 2827; Tel. +55 48 3721 2817
E-mail address: (A.L. Dafre)
Methods
Enzymatic analysis
Enzymatic analysis was performed on a Varian Cary 50 spectrophotometer (Agilent Tech.,CA, USA) following standard methods. In brief: GR was determined based on the NADPH absorbance decay due to GSSG reduction [1]. During the catalytic cycle, GPx uses GSH and cumene hydroperoxide as substrates and produces GSSG and the hydroperoxide alcohol. The GSSG formed is reduced by excess GR and NADPH added to the media. GPx activity is proportional to NADPH consumption [2].
Western blot
Striatal tissue was homogenized ( 1:10 w/v) in ice-cold lysis buffer (50 mM Tris-HCl pH 7.5, 1% Triton X-100, 100 mM NaCl, 5 mM EDTA pH 8.0, 40 mM β-glycerolphosphate, 50 mM NaF, 200 µM orthovanadate, 5% glycerol and protease inhibitors). The homogenates were centrifuged at 13000 g, at 4 ºC for 45 min. Prior to Western blot analysis, equivalent amounts of proteins were mixed in buffer (Tris 200 mM, glycerol 10 %, SDS 2 %, β-mercaptoethanol 2.75 mM and bromophenol blue 0.04 %), boiled for 5 min and kept at –20 ºC until analyses. Fifty micrograms of protein extract was subjected to SDS polyacrylamide gel electrophoresis (PAGE) using 12 % gels. Proteins were detected immunologically following electrotransfer onto nitrocellulose membranes (Amersham-Pharmacia Biotechnology, USA). Protein and molecular weight markers (bio-Rad, Mississauga, Canada) were revealed by Ponceau Red staining. The membranes were blocked (1 h) with 5 % skim milk in TBS (10 mM Tris, 150 mM NaCl, pH 7.5) and 0.05 % Tween-20. Blots were incubated overnight at 4°C with anti-TH antibody (sc-7847; Santa Cruz Biotechnology, USA) or anti-β-actin (sc-4778; Santa Cruz Biotechnology, USA) in TBS-Tween-BSA buffer (20 mM Tris base, 140 mM NaCl, 0.05 % Tween-20, 2 % BSA) and after that with horseradish peroxidase-conjugated anti-mouse IgG antibody for 1 h. Optical density (OD) of the Western blotting bands was quantified using the Scion Image® Software (Scion Corporation, Frederick, MD, USA). Values from TH bands were normalized with respect to β-actin bands.
2.2.4. Open-field test
The spontaneous locomotor activity was evaluated by the open-field test. Animals were individually placed in a wooden box (40 × 60 × 50 cm) with the floor divided into 12 rectangles. The number of squares crossed with all paws (crossing) was counted in a 5 min session. The apparatus was cleaned with a solution of 10 % ethanol between tests in order to hide animal clues.
2.2.5. Olfactory discrimination task
The olfactory discrimination ability was assessed with the olfactory discrimination task. The task was adapted from [3]. This task consisted of placing each mice for 5 min in a cage, which was divided into two identical compartments (30 × 30 × 20 cm) separated by an open door, so the animal could choose between one compartment with fresh sawdust (non-familiar) and another with unchanged sawdust (familiar) that the same mice had occupied for 48 h before the test. The time (seconds) spent by the rat in both compartments (familiar versus non-familiar) was counted.
Results
Tab. 2: Glutathione reductase (GR) and glutathione peroxidase (GPx) activity in the hippocampus and striatum of Mn- and curcumin-treated mice.
Group1 / Hippocampus / StriatumGR / GPx / GR / GPx
Ctl / 65.1 + 3.02 / 29.6 + 3.0 / 66.9 + 3.6 / 42.3 + 1.8
Cur500 / 62.7 + 6.2 / 31.2 + 2.6 / 68.1 + 7.3 / 43.6 + 4.2
Cur1500 / 66.1 + 7.1 / 31.3 + 8.4 / 68.0 + 6.5 / 38.7 + 7.0
Mn / 67.8 + 4.1 / 28.1 + 2.6 / 68.5 + 2.0 / 40.7 + 1.9
Mn+Cur500 / 64.4 + 5.6 / 33.7 + 3.9 / 72.1 + 2.4 / 44.9 + 6.1
Mn+Cur1500 / 64.6 + 0.5 / 31.5 + 2.1 / 70.8 + 7.0 / 45.9 + 5.9
1 No significant differences were detected by one-way Anova test (p > 0.05).
2Activity are presented as mean and standard error (N = 5-6) in nmol/min/mg of protein.
Supplementary Fig. 1. Object location task (A) and spontaneous locomotor activity assessed by the open field test (B) of Mn- and curcumin-treated mice. (A) The cognitive performance was analyzed by the relocation index (T shifted x 100) / (T + T shifted), where “T shifted” is the time of exploration of the familiar object that has been moved and T is the time spent exploring the familiar object (non-displaced). According to one-way Anova, no significant differences were found (N = 8-10). (B) In the open field test, no significant differences (N = 10-12) were found by the one-way Anova test (p > 0.05). Data are presented as mean and standard error.
Supplementary Fig. 2. Detection of tyrosine hydroxylase (TH) by Western blot in the striatum. No significant differences (N = 4) were detected by the one-way Anova test (p > 0.05).
References
[1] Carlberg I, Mannervik B. Glutathione reductase. Methods in Enzymology 1985;113:484–90.
[2] Wendel A. Glutathione peroxidase. Methods in Enzymology 1981;77:325–33.
[3] Prediger RDS, Batista LC, Takahashi RN. Caffeine reverses age-related deficits in olfactory discrimination and social recognition memory in rats. Involvement of adenosine A1 and A2A receptors. Neurobiology of Aging 2005;26:957–64.