PROTECTIVE EFFECTS OF KOLAVIRON AND QUERCETIN ON ATRAZINE-INDUCED NEURONAL AND TESTICULAR DAMAGE IN CELL LINES AND RATS

Abstract

Neurodegenerative diseases and decline in male fertility are reported to be associated with exposure to environmental chemicals. Many agrochemicals including atrazine are known to induce reproductive and neuronal toxicity. Kolaviron and quercetin are plant-derived flavonoids known to be protective against chemically-induced toxicity. The effects of kolaviron and quercetin against atrazine-induced neuronal and testicular toxicity were investigated. Forty male albino rats (weighing between 160 and 200 g) were divided into control, atrazine-treated (120 mg/kg), quercetin-treated (10 mg/kg) and atrazine plus quercetin-treated groups. Treatment periods were for 16 days by oral gavage. Sperm motility was evaluated by microscopy while lipid peroxidation and 17β-hydroxysteroid dehydrogenase (17β-HSD) were assayed by spectrophotometry. Testicular cells: Sertoli-germ cells (SGCs) and interstitial Leydig cells (ILCs) were isolated from the testis of peripubertal rats. SGCs were exposed In vitro to atrazine (232 uM) and quercetin (50 uM) alone or in combination for 6 and 24 h. The ILCs, pheochromocytoma (PC12) and human neuroblastoma (SH-SY5Y) cell lines were exposed to atrazine (232 uM) and kolaviron (60 uM) alone or in combination. Cells treated with 0.01% dimethylsulfoxide served as control. The effects of quercetin and kolaviron on cell viability were evaluated by the dimethylthiazol-tetrazolium bromide salt assay. The mRNA transcript levels of androgen receptor (AR), steroidogenic acute regulatory (StAR) protein and 3β-HSD were evaluated by real-time-PCR. Protein expression of apoptotic markers, Bax and Bel-2 were analysed by western blotting and expressed as integrated Density Value (IDV). Data were analysed using ANOVA and Student's t.test at p= 0.05. Sperm motility (91.7±4.9%) and 17β-HSD activity (5.2±1.0 Units/mg) were significantly higher while lipid peroxidation (234.0 ±24.9 nmol/mg protein) was lower in the control when compared with the atrazine-treated animals (30.2±5.7%, 2.7±0,9 Units/mg and 273.0±24.8 nmol/mg protein respectively). Quercetin co-treatment decreased these values by 120.0%, 74.0% and 36.0% respectively. Cell viability was significantly lower in atrazine-treated SGCs (85±10.1), SH-SY5Y (67.5%) and PC12 (79.2%) when compared with the respective controls (100.0±5.1, 100.0±18.4 and 100.0±9.1%). Co-treatment with quercetin improved SGCs viiability by 23.5% while kolaviron co-treatment increased SH-SY5Y, PC12 and ILCs viability by 18.5%, 20.2% and 34.3% respectively toward control level after 24h. The fold change in the expression of each target gene was expressed as 1 in the control. The relative gene expression of StAR and AR were higher in atrazine treated SGCs (1.9±0.1 and 5.4±0.1 respectively) when compared with control. Normalization in the expression of these genes to control value by 37.7% and 59.8% respectively was observed on co-treatment with quercetin after 6 h. Protein expression of Bax (1.2±0.03 IDV) was higher while Bel-2 (0.7±0.02 IDV) was lower in atrazine-treated SH-SY5Y and PC12 cell respectively when compared with control (0.9±0.02 IDV and 1.1±0.04). Normalization in the expressions of these proteins to control levels by 16.7% and 64.2% respectively was observed on co-treatment with kolaviron. Kolaviron protects against atrazine-induced toxicity in neuronal and interstitial Leydig cells. Quercetin prevented testicular damage caused by atrazine in rats and Sertoli-germ cells.