PROTECTIVE EFFECTS OF QUERCETIN AND RUTIN ON SULFASALAZINE-INDUCED TESTICULAR TOXICITY AND APOPTOSIS IN MALE WISTAR RATS

Abstract

Sulfasalazine is used as a therapy for inflammatory bowel diseases. High doses may be associated with male infertility; reversible only on drug withdrawal. Sulfasalazine metabolism generates oxidative stress as a mechanism associated with its toxicity. Quercetin and rutin are flavonoids which possess anti-oxidant properties and thus may mitigate against this effect. This study was designed to determine the protective potentials of quercetin and rutin on sulfasalazine-induced testicular toxicity. Seventy-two male Wistar rats (150-200 g) were divided into 6 groups (n=12) and treated orally with 0.5% carboxymethyl cellulose (control), quercetin (20 mg/kg), rutin (10 mg/kg), sulfasalazine (600 mg/kg), quercetin (20 mg/kg)+sulfasalazine (600 mg /kg) and rutin (10 mg/kg)+sulfasalazine (600 mg/kg) daily for 14 days respectively. Animals were sacrificed and blood, testes and epididymis obtained. Sperm motility, sperm count and acrosome reaction were assessed by light microscopy. Plasma testosterone, luteinizing hormone (LH) and follicle stimulating hormone (FSH) were determined by ELISA. Testicular 3B and 17B hydroxysteroid dehydrogenases (3B-HSB; 17B-HSD), and levels of lipid peroxidation (LPO) and reduced glutathione (GSH) in testis and semen were determined spectrophotometrically. Inducible Nitric Oxide Synthase (iNOS) and cyclooxygenase-2 expression were evaluated in testes and epididymis by immunohistochemistry. Caspase 3 and tumor suppressor p53 were assessed by ELISA and apoptosis by TUNEL assay in testes, semen and epididymis. Data were analysed using ANOVA at p=0.05. Sulfasalazine decreased sperm motility (66.7±5.8%), sperm count (103.7±8.1%) and acrosome reaction (42.5±2.9%) compared to control (88.3±11.6, 125.3±4.5, 92.5±2.9% respectively. Co-treatment of querccetin and rutin (20 mg/kg and 10 mg/kg respectively) with sulfasalazine showed no effect on sperm motility (70.0±10.0, 53.3±5.8%) and count (82.7±9.3, 76.3±2.5%) but increased acrosome reaction (85.0±5.0, 81.7±2.9%) relative to sulfasalazine traetment alone. Plasma LH and FSH (21.0±3.5, 28.0±2.8) U/L increased, while testosterone (0.1±0.0 U/L), testicular 3B-HSD and 17B-HSD (0.1±0.0 and 0.2±0.0 U/g) decreased in sulfasalazine-treated rats compared to control (FSH=3.5±0.7; LH=2.5±0.7, testosterone= 0.2±0.1) U/L and (3B-HSD=0.2±0.0;17B-HSD=0.3±0.0) U/g respectively. Quercetin and rutin decreased plasma LH (2.5±0.7) U/L and FSH (3.0±1.4, 8.5±2.1) U/L but increased testosterone (0.2±0.0, 0.4±0.1) U/L, testosterone 3B-HSD and 17B-HSD (0.2±0.0, 0.2±0.0, 0.6±0.0, 0.5±0.0) U/g respectively in sulfasalazine-treated rats. Levels of LPO (testes: 8.4±1.6; semen: 3.4±0.2) nmol/g increased while GSH (testes: 14.4±.0.5; semen: 54.5±1.3) ug/g decreased in sulfasalazine-treated rats versus control [LPO (testes: 4.6±0.6; semen; 2.5±0.2) nmol/g; GSH (testes: 17.5±1.7; semen: 71.6±2.6) nmol/g]. However, sulfasalazine increased p53 (7.4±1.3 pg/mL) and caspase 3 (3.4±1.2 pg/mL) above control levels (4.2±1.3, 1.9±0.0) pg/mL only in semen. Quercetin and rutin respectively decreased LPO levels (testes: 5.0±1.0, 3.3±0.7; semen: 3.0±.0.9, 2.8±0.1) nmol/g but increased GSM (testes: 16.4±1.8, 15.4±0.6; semen: 79.7±7.2. 74.8±7.4) ug/g in sulfasalazine-exposed rats. Caspase 3 and p53 decreased in semen of rats treated with sulfasalazine+quercetin(3.0+1.2, 5.1±0.7) pg/mL or rutin (3.3±0.5, 7.9±2.6) pg/mL. Expressions of iNOS, cyclooxygenase-2 and TUNEL positive sperm increased in epididymis of sulfasalazine-exposed rats. These were decreased in epididymis of rats when sulfasalazine was administered with quercetin or rutin. A high dose of sulfasalazine induced oxidative stress and apoptosis in testes and semen of rats. Co-administration of sulfasalazine with quercetin or rutin ameliorated these effects.