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

Doxorubicin (DOX) also known as adriamycin is an anthracycline antibiotic widely used in the treatment of several types of human ma- lignancies including haematological malignancies, solid tumors, soft- tissue sarcomas and breast carcinoma [1,2]. The therapeutic potential of DOX is achieved through the processes of intercalating into DNA, inhibiting topoisomerase II, preventing DNA and RNA synthesis [3]. However, the clinical use of DOX is limited due to the development of myocardial toxicity in patients as well as cytotoxic effects to normal cells, leading to unwanted side effects which may substantially impact patient health and quality of life [4,5]. In addition, nausea, vomiting, neutropenia, alopecia and arrhythmias are the acute adverse effects of DOX-therapy [6]. In connection with that, DOX-induced acute cardiotoxicity is approximately 11% during and within 2–3 days of its administration [7,8]. Metabolism of DOX in our body significantly in- creased the production of free radicals and the occurrence of lipid peroxidation [9,10] as well as depletion of antioxidants and sulfhydryl groups [11,12]. According to the present knowledge, the mechanisms responsible for DOX-induced cardiotoxicity appears to be multi- factorial, involving increased lipid peroxidation, oxidative stress, DNA/ RNA damage, endoplasmic reticulum-mediated apoptosis and dis- turbance of calcium homeostasis [13]. Additionally, the metabolism of DOX through NADPH-cytochrome P-450 enzyme leads to the formation of superoxide anions and hydroxyl radicals, which in turn cause injury to cellular membranes. Moreover, DOX can stimulate inducible nitric oxide synthase (iNOS) enzyme, increasing the production of nitric oxide (NO), which is associated with dilated cardiomyopathy and congestive heart failure [14]. Due to great importance of DOX in cancer che- motherapy, it is essential to reduce its toxicity to normal cells, a goal that can be achieved by concurrent administration of free radical scavenging agents such as antioxidants [15]. Thus, attenuating oxida- tive stress, inflammation and apoptosis is a potential therapeutic strategy against DOX-induced toxicity [16]. We therefore postulated that activation of endogenous antioxidant pathways might be a po- tential complement for DOX-induced toxicity. Thus, inhibition of apoptosis, attenuating oxidative stress as well as pro-inflammatory mediators, viz., nuclear factor-kβ (NFkβ), iNOS, cyclooxygenase-2 (COX-2) and interleukin-6 (IL-6) expression is an impending ther- apeutic approach against DOX-induced toxicity.

Indole-3-carbinol (I3C), a naturally occurring glucosinolate break- down product found in cruciferous vegetables such as cabbage, broccoli and Brussels sprouts [17,18]. It is one of the phytochemicals that was shown to poses potent anti-estrogenic [19] or apoptosis-inducing property in cancer cells [20]. Earlier studies reported that I3C has the ability to amplify some antioxidant enzymes activity such as hemox- ygenase-1 and glutathione transferase [21]. Moreover, I3C was shown to possess chemopreventive activity against benzo[a]pyrene-induced mouse forestomach carcinogenicity [22] as well as anti-tumor activities via interference with a variety of signal transduction pathways involved in cell survival [23]. Additionally, I3C attenuated lipid peroxidation by normalizing the activities of antioxidant enzymes in host organs. It was also reported that I3C has the ability to stabilized cell membrane and reduced leakage of myocyte injury marker enzymes [24]. Moreover, I3C modulated cell death mediators through down-regulating sphin- gosine kinase 1 (SphK1) activity and inflammatory mediators along with mitigated histological perturbations [14]. These facts motivated us to evaluate the protective role of I3C against DOX induced toxicities through inhibition of apoptosis, modulation of Nrf2/ARE and in- flammatory response pathway in Swiss albino mice. NMN

Materials and methods

Experimental animals

In this study, Adult (5–6 weeks old) Swiss albino female mice (25 ± 2 g b.w.), bred in the animal colony of Chittaranjan National Cancer Institute (Kolkata, India) were used. They were maintained at control temperature (23 ± 2 °C) and humidity (55 ± 10%) under al- ternating light and dark conditions (12 h/12 h). Animals were fed with standard food pellet diet (EPIC rat and mice pellet from Kalyani Feed Milling Plant, Kalyani, West Bengal, India) and drinking water was provided ad libitum. All procedures for animal experimentation used were approved by the Institutional Animal Ethics Committee (CPCSEA Reg. No.-1774/GO/RBi/S/14/CPCSEA, India)].

Chemicals

Indole-3-Carbinol was purchased from Sigma-Aldrich Chemicals Private Limited, Bangalore, India (Purity >96%). Doxorubicin was obtained from Cipla LTD, Verna, Goa, India. in situ cell death detection kit, AP was purchased from Roche Diagnostics India Private Limited, Bangalore, India. Nrf2, Keap1, HO1, NQO1, NFkβ(p50), iNOS, COX-2, IL-6, anti-mouse IgG-HRP, anti-goat IgG-HRP, anti-rabbit IgG-HRP and Luminol were bought from Santa Cruz Biotechnology (Texas, USA). GST-π was purchased from Cell Signaling Technology (Danvers, USA). GAPDH and Histone H3 were purchased from Novus Biologicals (Colorado, USA). All other chemicals not specified were obtained from Sigma-Aldrich Chemicals Private Limited, Bangalore, India and Merck (India) Limited, Mumbai, India.

Preparation and administration of I3C

Indole-3-Carbinol (I3C) was administered orally as a suspension using 5.5% propylene glycol in water. It was prepared each day just before treatment.

Median lethal dose (LD50) determination of I3C

The oral LD50 dose of the compound I3C was carried out as per the instruction by Organization for Economic Co-operation and Development (OECD) guidelines 425 by Up-and-Down-Procedure (UDP) [25].

 Experimental design for the toxicity study of the compound I3C

  • The animals were divided in to five groups containing six animals (n = 6) in each group.
    Vehicle-control group (VC): Each animal was orally treated with 5.5% propylene glycol in water for 28 days.
  • I3C (10 mg/kg b.w.)-treated group (I3C-10): I3C was given orally at the dose of 10 mg/kg b.w. for 28 days.
  • I3C (20 mg/kg b.w.)-treated group (I3C-20): I3C was given orally at the dose of 20 mg/kg b.w. for 28 days.
  • I3C (30 mg/kg b.w.)-treated group (I3C-30): I3C was given orally at the dose of 30 mg/kg b.w. for 28 days.
  • I3C (40 mg/kg b.w.)-treated group (I3C-40): I3C was given orally at the dose of 40 mg/kg b.w. for 28 days.

The mice were sacrificed 24 h after the last dose administration, on day 29.

Experimental groups for the evaluation of chemoprotective potential of I3C

In the present study, the animals were divided into five groups containing six animals (n = 6) in each group.

  • Vehicle control group (VC): Each animal was given 5.5% propy- lene glycol in water by oral gavages for 10 days.
  • Only I3C treated group (I3C): Each animal was orally treated with I3C at the dose of 20mg/kg b.w. throughout the experimental period (25 days).
  • DOX treated group (DOX): Each animal was intraperitoneally treated with only DOX in saline water (alternated days at a dose of 5 mg/kg b.w.) for up to day 9 (5 doses, resulting in a cumulative dose of 25 mg/kg b.w.).
  • DOX + I3C Concomitant-treated group (DOX + I3C Con): I3C was treated orally at the dose of 20 mg/kg b.w. from day 1 to day 10 and DOX was administered as DOX-treated group.
  • DOX + I3C Pre-treated group (DOX + I3C Pre): I3C was ad- ministered orally for 15 days prior to DOX treatment at the dose of 20 mg/kg b.w. and DOX was administrated as DOX treated group.
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