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

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LD50 of compound I3C

The oral LD50 of the compound I3C was found to be more than 2000 mg/kg b.w. (based on an assumed sigma of 0.5). Further, no sign of toxicity or moribund state were observed among the live animals during the entire duration of the study.

Effective dose selection of the compound I3C

Effective dose of I3C was determined on the basis of some toxicity and antioxidant efficacy parameters following 28 days treatment (Supplementary Table S1–S4). Treatment with I3C at 40 mg/kg b.w. increased the cardiac, hepatic and renal LPO level and serum ALT, AST and ALP activities. These findings rendered these doses to be toxic. However, treatment with I3C at 10, 20 and 30 mg/kg b.w. had no ad- verse effects on cardiac, hepatic and renal function markers. Whereas, amongst the four doses, I3C at 20 mg/kg b.w. showed most profound antioxidant efficacy indicated by its significant (P < 0.05) elevation of GSH level along with significant (P < 0.05) improved activities of other antioxidant enzymes and depletion of lipid peroxidation level. The organ sparing property of I3C was also confirmed by histological assessment (Supplementary Figure S1). Mice treated with I3C (10, 20 and 30 mg/kg b.w.) and vehicle control group showed normal archi- tecture, suggesting no detrimental changes or morphological dis- turbances. The overall cellular structure remains unaltered with no necrosis, infiltration or other significant lesions. Administration of I3C (40 mg/kg b.w.) showed mild swelling and inflammation in heart, liver and kidney tissues. 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.

(A) I3C decreased bone marrow ROS level after DOX-administration. Data were represented as mean ± SD, n = 6.*P < 0.05 significantly different from vehicle control (VC) group; #P < 0.05 significantly different from only DOX-treated group (one-way ANOVA followed by Tukey’s post hoc test). (B) Photomicrographs of femur section of mice after stain with hematoxylin and eosin (H&E × 400). Vehicle control (VC) group showed normal architecture of the femur; only I3C treated group (I3C) also showed normal morphology of the femur; only DOX-treated group (DOX) showed myelosuppressive effects i.e. myeloid hyperplasia and infiltration of the inflammatory cells; concomitant treatment with I3C (DOX + I3C Con) showed decreased hyperplasia and hemorrhage in femur; pretreatment with I3C (DOX + I3C Pre) showed marked improvement in the myeloid hyperplasia and hemorrhage in femur (H&E × 400).

(A) I3C decreased bone marrow ROS level after DOX-administration. Data were represented as mean ± SD, n = 6.*P < 0.05 significantly different from vehicle control (VC) group; #P < 0.05 significantly different from only DOX-treated group (one-way ANOVA followed by Tukey’s post hoc test). (B) Photomicrographs of femur section of mice after stain with hematoxylin and eosin (H&E × 400). Vehicle control (VC) group showed normal architecture of the femur; only I3C treated group (I3C) also showed normal morphology of the femur; only DOX-treated group (DOX) showed myelosuppressive effects i.e. myeloid hyperplasia and infiltration of the inflammatory cells; concomitant treatment with I3C (DOX + I3C Con) showed decreased hyperplasia and hemorrhage in femur; pretreatment with I3C (DOX + I3C Pre) showed marked improvement in the myeloid hyperplasia and hemorrhage in femur (H&E × 400).

Attenuation of ROS level

Intraperitoneal administration of DOX-significantly (P < 0.05) in- creased the bone marrow ROS level by 73.43% compared to vehicle control group (Fig. 1A). In contrast, oral administration of I3C in con- comitant and pretreatment schedule significantly (P < 0.05) depleted the bone marrow ROS level by 36.02% and 62.42% respectively com- pared to only DOX-treated group (Fig. 1A).

In case of cardiac tissue, intraperitoneal administration of DOX- significantly (P < 0.05) elevated the cardiac whole cell, mitochondrial and cytosolic ROS level by 76.51%, 88.58% and 68.82% respectively compared to vehicle control group (Fig. 5A). Whereas, oral adminis- tration of I3C in concomitant treatment schedule significantly (P < 0.05) depleted the cardiac whole cell, mitochondrial and cyto- solic ROS generation by 29.77%, 43.18% and 18.07% respectively compared to only DOX-treated mice. Additionally, pretreatment with I3C most significantly (P < 0.05) depleted the ROS level in cardiac whole cell, mitochondrial and cytosolic ROS level by 54.95%, 70.78% and 39.73% respectively compared to only DOX-treated mice. Bioavailable NMN

Reduction of NO level

The level of cardiac NO in DOX-treated group was significantly (P < 0.05) increased by 52.91% compared to vehicle control group (Fig. 5B). In contrast, oral administration of I3C in concomitant and pretreatment schedule significantly (P < 0.05) decreased the NO level by 24.45% and 40.14% respectively compared to only DOX-treated group.

Preventive effects of I3C against DOX-induced genotoxicity

Inflection of bone marrow cell proliferation
Intraperitoneal administration of DOX-significantly (P < 0.05) de-

creased the bone marrow cell proliferation by 62.14% compared to vehicle control group (Fig. 2). However, oral administration of I3C significantly (P < 0.05) reversed the inhibitory effect of DOX on cel- lular proliferation and raised BrdU LI to 41.77% and 54.00% respec- tively in concomitant and pretreatment schedule compared to only DOX-treated mice.

Alleviation of chromosomal aberration

DOX-administration significantly (P < 0.05) increased the pro- portion of chromosomal aberrations by 74.79% compared to vehicle control group (Table 4; Fig. 3A–B). The nature of chromosomal aber- rations observed was chromosome fragments (CF), breaks (B), ring formation (R), stretching (STR), end to end union (EEU), centric fusion (CF) etc. On the other hand, administration of I3C in concomitant and pretreatment schedule significantly (P < 0.05) reduced the chromo- somal aberration by 32.37% and 54.76% respectively compared to only DOX-treated group.

I3C mediated protection against DOX-induced myelosupression. Cell proliferation assay showing non-proliferating cells and proliferating cells (indicated by BCIP/NBT staining), ×400 magnification, scale bar = 50 μm. Dark-stained cell nucleus represents proliferating cells; unstained cells represent non proliferating cells. The quantification of the BrdU LI (%) were calculated for each group and data were represented as mean ± SD, n = 6. *Significantly (P < 0.05) different from vehicle control (VC) group and #P significantly (P < 0.05) different from only DOX-treated group (DOX) (one-way ANOVA followed by Tukey’s post hoc test). DOX-administration significantly depleted the proportion of proliferating cells compared to vehicle control (VC) group. In contrast, oral administration of I3C significantly (P < 0.05) increased the percentage of proliferating cells compared to the DOX-mono- therapy.

I3C mediated protection against DOX-induced myelosupression. Cell proliferation assay showing non-proliferating cells and proliferating cells (indicated by BCIP/NBT staining), ×400 magnification, scale bar = 50 μm. Dark-stained cell nucleus represents proliferating cells; unstained cells represent non proliferating cells. The quantification of the BrdU LI (%) were calculated for each group and data were represented as mean ± SD, n = 6. *Significantly (P < 0.05) different from vehicle control (VC) group and #P significantly (P < 0.05) different from only DOX-treated group (DOX) (one-way ANOVA followed by Tukey’s post hoc test). DOX-administration significantly depleted the proportion of proliferating cells compared to vehicle control (VC) group. In contrast, oral administration of I3C significantly (P < 0.05) increased the percentage of proliferating cells compared to the DOX-mono- therapy.

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