Eight-weeks-old male B6C3 mice (25–30 g) were divided into five groups of six mice each and maintained on a 12 h light/dark cycle in a temperature-controlled (23±2 °C) room. The mice had free access to food and water. All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of Kentucky.
Mice were treated with a single dose of 20 mg/kg of Doxorubicin-Adriamycin (Dox) (DOXOrubicin HCl, Bedford Laboratories, Inc., Bedford, OH, USA) via i.p. injection. Xanthone (200 mg/kg, Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 2% Gum acacia (Sigma-Aldrich, St. Louis, MO, USA) and administered as i.p. injection 15 min prior to the Dox treatment. Mice were euthanized 72 h after treatment. The experiments were conducted with five independent groups (n
=6 per group) as follows:
- Group 1: Control (Saline)
- Group 2: Dox (20 mg/kg)
- Group 3: Vehicle group (2% Gum acacia)
- Group 4: Xanthone (200 mg/kg)
- Group 5: Xanthone (200 mg/kg) + Dox (20 mg/kg)
Animals were anesthetized using Nembutal\sodium solution (65 mg/kg) (Abbott Laboratories, North Chicago, IL, USA) and blood was obtained via left ventricle puncture. Serum was collected for the macrophage stimulation experiments. Brain tissues were dissected from animals in all treatment groups and placed in ice-cold lysing buffer containing 4 g/ml leupeptin, 4 g/ml pepstatin, 5 g/ml aprotinin, 2 mM ethylenediaminetetraacetic acid (EDTA), 2 mM ethylene glycol-bistetraacetic acid (EGTA), and 10 mM 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES), pH 7.4. The brain tissues were homogenized by 20 passes of a Wheaton tissue homogenizer, and the resulting homogenate was centrifuged at 20,000 g for 10 min and then assayed for protein concentration by the Pierce BCA method (Bradford, 1976
Macrophage stimulation and cytokine assay
For experiments with macrophages, the mouse macrophage cell line J774A.1 (American Type Culture Collection) was cultured in DMEM supplemented with 10% (vol/vol) FBS, streptomycin (100 µg/ml) and penicillin (100 U/ml). All cultures were incubated at 37 °C in a humidified atmosphere with 5% CO2. The cells were seeded onto a 48-well plate at 5 × 105 cells/well and allowed to grow overnight. Serum collected from the respective groups was inactivated at 56 °C for 30 min and filtered through 0.22 µm membrane and added to the cell cultures. After 24 h, incubation media were collected and TNFα levels were measured with an enzyme-linked immunosorbent assay (mouse TNF-alpha, R&D Systems, Minneapolis, MN, USA) following the manufacturer’s instructions. The TNFα concentration in the sample was calculated from a recombinant mouse TNFα standard curve. The cells were also processed and measured for protein carbonyls 3-NT (nitrotyrosine) and 4-HNE (hydroxynonenal), as described below.
Measurement of protein carbonyls
Each sample (5 µl) of brain homogenate and macrophage cell lysate was mixed with 12% sodium dodecyl sulfate (SDS; 5 µl) and 10 µl 1:10 diluted 2,4-dinitrophenylhydrazine (DNPH) from 200 mM stock and incubated at room temperature for 20 min, followed by neutralization with 7.5 µl neutralization solution (2 M Tris in 30% glycerol). Protein (250 ng) was loaded into each well on a nitrocellulose membrane using a slot blot apparatus connected to a vacuum source. The membrane was blocked in blocking buffer (3% bovine serum albumin in PBS containing 0.01% (w/v) sodium azide and 0.2% (v/v) Tween 20) for 1 h and incubated with a 1:100 dilution of anti-DNP polyclonal antibody in PBS containing 0.01% (w/v) sodium azide and 0.2% (v/v) Tween 20 for 1 h. After incubation with the primary antibody, the membrane was washed three times in PBS at 5-min intervals then incubated for 1 h with an anti-rabbit IgG alkaline phosphatase secondary antibody diluted in PBS in a 1:8,000 ratio. The membrane was washed three times in PBS for 5 min and developed in substrate solution prepared with Sigma fast tablets (5-bromo-4-chloro-3-indolyl phosphate [BCIP]/nitro blue tetrazolium [NBT] substrate). The membranes were dried, scanned with Adobe Photoshop software, and quantified in Scion Image program (PC version of Macintosh-compatible NIH Image).
Measurement of 4-HNE and 3-nitrotyrosine (3-NT)
Each sample (5 µl) of brain homogenate and macrophage cell lysate was mixed with 12% SDS (5 µl) and 5 µl modified Laemmli buffer containing 0.125 M Tris base, pH 6.8, 4% (v/v) SDS, and 20% (v/v) glycerol, incubated for 20 min at room temperature and loaded (250 ng) into each well on a nitrocellulose membrane in a slot blot apparatus connected to a vacuum source. The membrane was treated as described above except that 1-h primary antibody incubation was replaced with a 90 min incubation with a 1:5,000 dilution of anti-HNE polyclonal antibody in PBS.
Each sample (5 µl) of brain homogenate was also mixed with 12% SDS (5 µl), and modified Laemmli buffer (5 µl) containing 0.125 M Tris base, pH 6.8, 4% (v/v) SDS, and 20% (v/v) glycerol, incubated for 20 min at room temperature and loaded (250 ng) into each well on a nitrocellulose membrane in a slot blot apparatus under vacuum. The membrane was treated as described above except that a 1:2,000 dilution of anti-3-nitrotyrosine polyclonal antibody was used for the primary antibody incubation.
Western blot analysis
Perfused brain homogenates were separated using 12.5% denaturing polyacrylamide gel electrophoresis (SDS–PAGE) and transferred onto a nitrocellulose membrane. The membrane was blocked for 1 h at room temperature in blocking solution consisting of 5% nonfat dried milk, 0.5% Tween-20, and Tris-buffered saline (TBST), pH 7.9. After blocking, the membrane was incubated overnight at 4 °C with primary antibodies against TNF (Upstate, Lake Placid, NY, USA), inducible nitric oxide synthase (iNOS), Bax, Bcl-xL (Santa Cruz Biotechnology, Santa Cruz, CA, USA), p53 (Cell signaling, Danvers, MA, USA) and β-actin (Clone AC-74, A5316, Sigma, St. Louis, MO, USA). The membrane was washed twice in TBST and incubated for 1 h with horseradish peroxidase-conjugated secondary antibodies in blocking solution. After incubation with secondary antibodies, the membrane was washed twice with TBST and once in TBST without 0.5% Tween-20. Immunoreactivities of the protein bands were detected by enhanced chemiluminescence autoradiography (ECL, Amersham Pharmacia Biotech, Arlington Heights, IL, USA) as described by the manufacturer.
Brain nitric oxide levels
Nitric oxide (NO) production was measured based on the Griess reaction (Fox et al., 1982
). A final volume of 200 µl, including 40 µl of sample, 40 µl of nitrate/nitrite assay buffer, and 10 µl each of nitrate reductase cofactor and nitrate reductase enzyme preparation, was incubated covered, at room temperature, for 3 h. After the addition of 50 µl of Griess reagents, the samples were kept at room temperature for 10 min and formation of a deep purple azo compound was detected using a spectrophotometer at 540 nm. All reagents were obtained from Cayman Chemical (Ann Arbor, MI, USA).
Caspase 3 activity assay
Caspase 3 activity assay was performed with the use of a colorimetric substrate in accordance with the protocol supplied by the manufacturer (Sigma, St. Louis, MO, USA). In brief, mice were anesthetized and perfused with 1× PBS to reduce any enzyme activity associated with intravascular blood components. Brain was dissected and homogenized in lysis buffer containing 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.4, 5 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 5 mM Dithiothreitol (DTT) plus 1 µg/ml aprotinin and pepstatin. The brain homogenates were incubated on ice for 15 min and centrifuged at 16,000×g for 10 min. The protein concentration was determined by the Bradford method and the caspase 3 activity in the supernatant was measured immediately. 50 µg protein samples in 10 µl were added to 980 µl assay buffer. The reaction was initiated by adding 10 µl of 20 mM of the caspase 3 substrate Ac-DEVD-pNA. The tubes were covered and incubated at 37 °C overnight. Cleavage of the chromophore from the substrate was detected spectrophotometrically at a wave-length of 405 nm.
The assay was performed following the manufacturer’s instructions (Promega, Madison, WI, USA). Briefly, the cryosections of brain were fixed with 4% paraformaldehyde, permeabilized with Triton X-100, and incubated with biotinylated nucleotide and recombinant termination deoxynucleotidyltransferase (rTdT) for 1 h at 37 °C. The fragmented DNA labeled at the ends was coated with horseradish peroxidase-labeled streptavidin (streptavidin HRP) and detected as dark brown condensed nuclei, a positive indication of cell death. The sections were counterstained with Methyl-Green by incubating 5 min in Methyl Green staining dye followed by repeated rinsing in distilled water and subsequent quick dehydration using 95% alcohol (10 dips) and two changes of 100% alcohol (10 dips each). The sections were rinsed finally in xylene and mounted with mounting medium. Positive control samples were prepared by incubating sections with DNase I prior to treatment with terminal transferase. Negative controls consisted of specimens in which deoxynucleotidyltransferase were omitted.
Statistical analyses were performed using one-way ANOVA followed by Newman–Keuls post-test (GraphPad Prism-4). A P-value of less than 0.05 was considered a significant difference.