Protective Effect of Thymoquinone against Cyclophosphamide-Induced Hemorrhagic Cystitis through Inhibiting DNA Damage and Upregulation of Nrf2 Expression

Abstract

Cyclophosphamide (CYP) induced hemorrhagic cystitis is a dose-limiting side effect involving increased oxidative stress, inflammatory cytokines and suppressed activity of nuclear factor related erythroid 2-related factor (Nrf2). Thymoquinone (TQ), an active constituent of Nigella sativa seeds, is reported to increase the expression of Nrf2, exert antioxidant action, and anti-inflammatory effects in the experimental animals. The present study was designed to explore the effects of TQ on CYP-induced hemorrhagic cystitis in Balb/c mice. Cystitis was induced by a single intraperitoneal injection of CYP (200 mg/kg). TQ was administered intraperitoneally at 5, 10 and 20 mg/kg doses twice a day, for three days before and three days after the CYP administration. The efficacy of TQ was determined in terms of the protection against the CYP-induced histological perturbations in the bladder tissue, reduction in the oxidative stress, and inhibition of the DNA fragmentation. Immunohistochemistry was performed to examine the expression of Nrf2. TQ protected against CYP-induced oxidative stress was evident from significant reduction in the lipid peroxidation, restoration of the levels of reduced glutathione, catalase and superoxide dismutase activities. TQ treatment significantly reduced the DNA damage evident as reduced DNA fragmentation. A significant decrease in the cellular infiltration, edema, epithelial denudation and hemorrhage were observed in the histological observations. There was restoration and rise in the Nrf2 expression in the bladder tissues of mice treated with TQ. These results confirm that, TQ ameliorates the CYP-induced hemorrhagic cystitis in mice through reduction in the oxidative stress, inhibition of the DNA damage and through increased expression of Nrf2 in the bladder tissues.

Uroprotective mechanisms of natural products against cyclophosphamide-induced urinary bladder toxicity: A comprehensive review

“…In models of CYP-induced urotoxicity, DADS treatment exhibited a notable upregulation of bladder Nrf2/HO-1/NQO-1 protein expression (Kim et al, 2014) and a similar result was observed with a medicinal plant derived from Nigella sativa seeds (F: Ranunculaceae) called thymoquinone which possessed an antioxidant activity and showed a marked rise in the bladder protein expression of Nrf2 (Gore et al, 2016).…”

“…Kim et al (2014) reported that CYP induced a marked upregulation of bladder Nrf2/HO-1/NQO-1 protein expression reporting that urotoxicity induced Nrf2 with concomitant phase II enzymes as a cytoprotective mechanism (Kim et al, 2014). A contradictory study showed decreased bladder protein expression of Nrf2 accompanied with CYP injection and explained this action through CYP metabolites, which diminish the Nrf2 activity and subsequently inhibit phase II antioxidant enzymes resulting in increased oxidative tissue damage (Gore et al, 2016). These controversial results could be attributed to different study designs including the difference in the CYP dose and the experimental animals used.…”

“…Three assays were used to detect DNA fragmentation including; DNA ladder assay detecting the DNA fragment pattern, comet assay or single cell gel electrophoresis assay detecting the DNA damage in a single cell, and terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay which detected the apoptotic cells that undergo severe DNA degradation throughout the apoptosis late stages (Kyrylkova et al, 2012). In the comet assay, tail length, the migration extent of the DNA, and the percentage of the DNA content in the tail comet were shown to be related to the rate of the DNA breakage (Gore et al, 2016).…”

Hepatoprotective and antiapoptotic role of aged black garlic against hepatotoxicity induced by cyclophosphamide

“…Zhang et al (2012) proved that the CP produced significant DNA damage and cell apoptosis in mouse bone marrow cells and peripheral blood leukocytes, DNA damage, and cell apoptosis were investigated by single cell gel electrophoresis, flow cytometry assay with annexin V-FITC/propidine iodide (PI), and acridine orange (AO)/ethidium bromide (EB) staining assay. Gore et al (2016) investigated the effect of CP on bladder genomic DNA by agarose gel electrophoresis in mice, and they found that genomic DNA samples isolated from bladders of mice treated with cyclophosphamide showed significantly greater DNA fragmentation. Cell apoptosis is involved in the CP-induced acute hepatotoxicity, characterized by activation of immunohistochemical marker caspase-3, a key cell death protease (Li et al, 2015).…”

Therapeutic Potential and Pharmaceutical Development of Thymoquinone: A Multitargeted Molecule of Natural Origin

“…TQ treatment administered intraperitoneally in the doses of 5, 10, and 20 mg/kg before and after the cyclophosphamide injections prevented histological perturbations as evidenced by decreased cellular infiltration, edema, epithelial denudation and hemorrhage in the bladder tissue, reduced oxidative stress, lipid peroxidation and inhibited DNA fragmentation. TQ also induced Nrf2 expression in the bladder tissues of mice concomitant to the restored anti-oxidant s in the bladder tissues (Gore et al, 2016 ).…”