INFLUENCE OF KOLAVIRON ON TESTICULAR DAMAGE AND IMPAIRED STEROIDOGENESIS INDUCED BY ETHYLENE GLYCOL MONOETHYL ETHER AND CARBENDAZIM IN RATS

Abstract

The reproductive toxicities of ethylene glycol monoethyl ether (EGEE), a widely used industrial solvent, and carbendazim (CBZ), a fungicide, are known in experimental animals. However, the molecular mechanisms involved are not well known. The mammalian testis, which produces germ cells for spermatogenesis and steroidogenesis, is sensitive to oxidative stress which has been linked to apoptosis. Kolaviron, a biflavonoid from Garcinia kola seed, is well reported to possess antioxidant and antigenotoxic properties. The effects of EGEE and CBZ on antioxidant system, stress-inducible proteins, steroidogenesis and apoptosis-related proteins as well as the protective role of Kolaviron in EGEE- and CBZ-induced testicular damage were investigated in rats. Adult male Wistar rats were orally exposed to EGEE (200mg/kg) and CBZ (200mg/kg) singly or in combination with either kolaviron (100 and 200 mg/kg) or vitamin E (50mg/kg) (a standard antioxidant). EGEE and CBZ treatments lasted 14 days and 24 hours, respectively. Following exposure, biomarkers of oxidative injury and the activities of testicular 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase (17β-HSD) were quantified spectrophotometrically whereas plasma Ievels of luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone, prolactin, triiodothyronine and thyroxine were determined by ELISA. Testicular levels of steroid acute regulatory (StAR) protein and androgen binding protein (ABP), stress-indelible proteins [clusterin (sCLU) and heat shock proteins (HSP)], opoptosis-related proteins, namely cytochrome c, caspase-3 and caspase-9, Fas and Fas-L as well as expression of nuclear factor kappa B (NF- kB) were assessed by western blotting. Localization of caspase-3 and fa in testes was confirmed by Immofluorescence staining. Testicular damage was confirmed by light microscopy and germ cell apoptosis was determined by terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate in situ-nick-end-labeling (TUNEL) technique. Data were analyzed using ANOVA and Student t-test (p=0.05). EGEE and CBZ treatments disrupted testicular antioxidant system, increased lipid peroxidation, but significantly decreased StAR and ABP levels and 3β-HSD and 17β- HSD activities EGEE-exposure, alone, elicited significant increase in the levels of inducible HSP (4.0-fold) and sCLU (2.0-fold) whereas it significantly diminished LH (29%), testosterone (44%), prolactin (81%), triiodothyronine (64%) and thyroxine (44%) levels with marked elevated FSH (50%) level. The increased expression of active caspase-3 (4.2-fold), caspase-9 (2.8-fold), Fas (3.1-fold) and Fas-L (3.0-fold) was accompanied by NF-kB down-regulation (2.5-fold) and translocation of cytochrome c (3.0-fold) from mitochondria to cytosol in the testis. Also, increased caspase-3 and Fas levels in testicular sections from immunofluorescence staining were consistent with the increased TUNEL-positive nuclei and marked testicular degeneration observed in both treatments Kolaviron and Vitamin E significantly ameliorated testicular atrophy and alterations in antioxidant status and steroidogenes is induced by EGEE and CBZ. While kolaviron at 100 and 200 mg/kg inhibited testicular apoptosis by 88% and 86% in EGEE-treated rats and by 91% and 93% in CBZ-treated rats respectively, inhibition by vitamin E were 88% and 96% in EGEE-and CBZ-treated rats respectively. Ethylene glycol monoethyl ether and carbendazim induced testicular oxidative damage and impaired steroidogenesis in rats. Kolaviron and vitamin E by virtue of their antioxidant and antiapoptotic properties inhibited ethylene glycol monoethyl ether- and carbendazim-induced testicular toxicity in rats .