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Prevention of doxorubicin (DOX)-induced genotoxicity and cardiotoxicity

The mice were sacrificed on day 11 and the parameters described below were studied.

Doxorubicin dose was selected on the basis of (a) when DOX used in combination with other chemotherapy drugs for the treatment of dif- ferent type of cancer, the most commonly used dosage of doxorubicin is 60–75mg/m2 IV once every 21 days. This dosage is equivalent to 20–25 mg/kg b.w. in mice (b) DOX in this clinically relevant dose is previously reported for its cardiotoxicity in mice [26,27]. On the basis of above back ground doxorubicin dose was selected at 25 mg/kg b.w. Dose of I3C was selected on the basis of some toxicity and antioxidant efficacy parameters following 28 days treatment. The results showed that I3C at 20 mg/kg b.w. is safe and most efficacious (Supplementary Table S1–S4). The organ sparing property of I3C was also confirmed by histological assessment (Supplementary Figure S1). So, on the basis of safety and efficacy end points, the oral dose of I3C at 20 mg/kg b.w. was selected for further preclinical study. In the present study we evaluate the protective role of I3C against DOX- induced genotoxicity and cardiotoxicity in Swiss albino mice. For this purpose, we adminis- tered I3C along with DOX in concomitant and 15 days pre-treatment schedules to find out any schedule-dependent difference in efficacy as well as to find out whether the compound I3C can provide any added protection to the target organs/cells before exposure to the che- motherapeutic agent DOX.

Sample collection

Before euthanasia, all animals were fasted for 4 h then retro-orbital venous plexus technique were used to collect the blood samples. The blood samples were divided in to two micro centrifuge tubes. One set of tubes were left to clot at room temperature and serum were separated

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. Nicotinamide Mononucleotide

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.

Clinical toxicity markers

Cardio toxicity markers namely serum creatine phosphokinase (CPK) and creatine kinase myocardial bound (CK-MB) activity and some other clinical toxicity markers i.e. serum ALT, AST, ALP activity by centrifugation at 2000 × g for 15 min and further analysis.

Preparation of bone marrow cell suspension 

The bone marrow cells from both femurs were collected for studies various genotoxicity parameters. The mice bone marrow from the fe- murs was flushed out with 1 × phosphate buffer saline (PBS) into a centrifuge tube. The cells were centrifuged at 500 g for 10 min and pellets were re-suspended in, as little supernatant as possible. Then the bone marrow cellularity was determined and the samples were pro- cessed according to the methods described below.