Prevention of doxorubicin (DOX)-induced genotoxicity and cardiotoxicity: Effect of plant derived small molecule indole-3-carbinol (I3C) on oxidative stress and inflammation

Determination of reactive oxygen species (ROS) generation

The intracellular amounts of reactive oxygen species (ROS) were measured in bone marrow cells and in whole cell, mitochondrial and cytosolic fraction of cardiac tissue homogenate by a fluorometric assay with DCFH-DA [28]. Briefly, bone marrow cells (2 × 106) and cardiac tissue homogenate (homogenized in Locke’s Buffer, pH 7.4) were loaded with 10 μM DCFH-DA and incubated in dark for 30 min and 45 min respectively to allow the formation of DCF and then analyzed for fluorescence (excitation 485 nm/emission 529 nm) using spectro- fluorimeter (Varian Cary Eclipse). Values were expressed as fluores- cence intensity normalized/mg protein.

Measurement of cardiac nitrite production

Nitric oxide (NO) production in heart tissue homogenate was de- termined by estimating the level of stable NO metabolites, viz., nitrate (NO3−) and nitrite (NO2−) ions [29]. The total nitrite level was de- termined by reaction with Griess reagent (1% sulphanilamide, 5% phosphoric acid and 0.1% NEDD) using sodium nitrite as standard by UV–vis spectrophotometer (Infinite® 200 PRO; TECAN). The absor- bance was taken at 545 nm and expressed as μM nitrite/mg protein.

Genotoxicity studies

In situ cell proliferation

Bone marrow cell proliferation was measured by using BrdU Labeling and Detection Kit II [30]. Briefly, the bone marrow cells were placed into BrdU-added pre warmed (37 °C) cell culture medium for 60 min at 37 °C, 5% CO2 where the DNA of proliferating S-phase cells get labeled with BrdU. Then the cells were smeared on slides and fixed. The slides were subsequently incubated with an anti-BrdU monoclonal antibody (37 °C for 30 min), which was detected by an alkaline phos- phatase conjugated-anti mouse-immunoglobulin antibody (anti-mouse- Ig-AP). The bound anti-mouse-Ig-AP was visualized using BCIP/NBT, an AP-substrate solution. The slides were then analyzed with a light mi- croscope (DM1000; Leica). The BrdU labeling index (BrdU LI) was de- termined by dividing the number of labeled cells by total number of cells counted and then multiplying by 100.

Chromosomal aberration study

Cells arrested in metaphase plate were examined microscopically for structural chromosome aberrations. 90 min before sacrifice, mice were intraperitoneally injected with 0.03% colchicines (1 ml/100 g b.w.). Marrow of the femur was flushed in 1% sodium citrate (37 °C) and fixed in acetic acid/methanol (1:3) solution. Slides were prepared by the conventional flame drying technique followed by Giemsa staining (1: 5 dilutions in Sorenson’s phosphate buffer). The slides were examined at 1000 × magnification (DM1000, Leica) for scoring chro- mosomal aberration (CA) [31].

Micronucleus assay

0.075 M KCl solutions were used to collect mice bone marrow cells and collected cells were incubated at 37 °C for 10 min. Then the tubes were centrifuged at 500 × g for 10 min and two smears of bone marrow were prepared from each mouse and stained by Giemsa (1:5 dilutions in Sorenson’s phosphate buffer) [32]. The slides were examined for the presence of micronucleus at 1000 × magnification (DM1000, Leica).

Measurement of extent of DNA damage by comet assay

DOX-induced possible DNA damage was evaluated by using alkaline single cell gel electrophoresis or comet assay technique [28]. The mi- croscope slides were carefully dried at room temperature and stained with ethidium bromide in distilled water (20μg/ml; 80μl/slide). The slides were examined at × 400 magnification under a fluorescence microscope (DM4000 B; Leica) with imaging system. Komet 5.5 soft- ware (Andor Technology) was used to take the photomicrograph of cells and to analyze various parameters of the comet.

Damage cell (%) = Number of damage cells × 100 Total number of cells counted

In situ cell death (apoptosis)

Apoptosis of bone marrow cells and cardiac tissues were determined by using in situ cell death detection kit, form Roche Diagnostics, USA according to the manufacturer’s instructions. Briefly, the bone marrow cells were smeared on slides, permeabilized using 0.1% Triton X-100 and the slides were incubated with TUNEL reaction mixture containing the TdT and fluorescein-dUTP, at 37 °C for 60 min in a humidified chamber. The slides were then analyzed under a fluorescence micro- scope (DM4000 B, Leica) and photomicrographs were taken at × 400 magnification. The apoptotic cells were identified by green fluores- cence. Whereas, in case of cardiac tissues, the slides were permeabilized using 0.1% Triton X-100 and the slides were incubated with TUNEL reaction mixture containing TdT and fluorescein-dUTP, at 37 °C for 60 min in a humidified chamber. The slides were further treated with antifluorescein antibody conjugated with alkaline phosphatase (37 °C for 30 min in a humidified chamber). The bound alkaline phosphatase was then stained with BCIP/NBT and observed under light microscope (DM1000, Leica, Germany). Apoptotic index (AI) was determined as the percentage of the labeled nuclei with respect to the total number of nuclei counted. Sublingual NMN

DOX-induced cardiotoxicity

Haematological parameters

Blood hemoglobin (Hb) level was determines according to the Sahli’s method. Haematological parameters i.e., RBC, WBC and Differential WBC count were performed by standard procedure [33,34]. Bone marrow cellularity, Spleen cell count and thymus cell count were also determined by standard procedure.

 




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