Study On Fluorochlondone Testicular Toxicity And Its Mechanisms

Fluorochloridone(3-chloro-4-(chloromethyl)-1-[3-(trifluoromethyl)= phenyl]-2-pyrrolidinone, FLC) is a ketopyrrolidine herbicide widely used in Europe and the US. It has also been used in as temporary registrated product in China Pesticide Information Network (ICAMA). Literature reported few FLC toxicity studies. Two abstracts published in JOURNAL OF ANDROLOGY pointed out that FLC caused testes atrophy, sperm quality and quantity reduction, serum FSH level increase and reproductive function decline in rats. By the end of 2010, the Europe Food Safety Authority (EFSA) noted in a FLC risk assessment report that FLC had potential toxicity in male reproductive system. This toxicity mainly targets testes and epididymis, producing decline in sperm counts and quantity as well as Sertoli cell vacuoles. FLC was also classified as a possible environmental endocrine disruptor by the Health and Consumer Council of European Union in a Conference March 2011. In this study, we aimed to assess the impact of FLC on SD rat testes, FLC induced germ cells and its possible mechanisms, and Sertoli cell injuries aroused by FLC. Three research models including male SD rats exposed to FLC in vivo, primary Sertoli-germ cell co-culture system and primary Sertoli cell in vitro were applied in this research. It aimed to investigating FLC testicular toxicity and its main signaling pathway mechanisms, so as to provide basic data and reference for FLC health risk assessment, moreover to draw guidance for the future understanding of the reproductive toxicity mechanisms of herbicides with similar structure.Impact of FLC on rat testes40 Male SD rats were assigned randomly into a control group and 3 FLC treatment groups. These groups, each of 10 male rats, were given FLC by gavage at the dose levels of 0 (control),30,150, and 750mg/kg bw/d,7 days/week for 28 days. FLC testicular toxicity evaluation was operated by using testicular histopathology examination, seminiferous tubules electron microscope observation, epididymal cauda sperm count and serum testosterone (T) detection after treatment period. Oxidative stress biomarkers of oxidative stress including MDA, GSH, SOD, CAT, GSH-Px, GSH-GR and GSH-ST in testes were detected by commercial kits.1. FLC disrupted oxidation and anti-oxidation balance in testes. MDA level, the lipid peroxidation marker, was significantly increased in the medium and higher dose groups and GSH level was significantly declined in each treatment group compared with control (p<0.05 or p<0.001). Anti-oxidative enzymes activity compromise SOD, CAT, GSH-Px, GSH-GR and GSH-ST in higher dose group were statistically reduced under the influence of FLC (p<0.05 or p<0.001). There were a significant decrease of all the above anti-oxidative enzymes activity (p<0.05 or p<0.001) except GSH-GR in the medium dose group. The SOD and CAT activity were also significantly decreased in the lower dose group.2. FLC caused testes in higher dose group had statistical reductions in testes weights, testes organ coefficient, epididymis weights, serum T level and cauda epididymal sperm counts compared with control (p<0.05 or p<0.001). The rats in medium and higher dose group had histopathological injuries in testes including interstitial edema, tubular architecture disorder, epithelial cell disruption, germ cells layer damage, sperm cells disappear, Sertoli cells vacuolization, nuclear integrity damage. Seminiferous tubules electro microscope observation displayed fragmented germ cells, abnormal germ nucleolus and mitochondrial structure.95% lower confidence limit of benchmark doses for testes weights, testicular organ coefficient, epipdidymal weights, cauda epididymal sperm counts, serum testosterone and testicular histopathological injuries were 65.7,76.0,23.0,2.4,99.9 and 12.9 mg/kg bw/d respectively. Thus, cauda epididymal sperm count and testicular pathological injuries were most sensitive for FLC exposure.FLC induced primary co-cultured Sertoli-germ cell apoptosis and its possible signaling pathway mechanisms.Testes of 28-30-day old SD rats were aseptically removed and directed two steps of enzyme digestion were used for the isolation of Sertoli-germ cells. A 0.1% DMSO solvent control group and three FLC exposure groups (10-8,10-7 and 10-6M) were selected, and experimental results suggested that:1.MTT assay and detached germ cells counts by hemocytometer were performed. Relative Cell viability declined to 84.76±4.91 (%) in higher dose group after 6 h exposure and further decreased to 72.16±2.53,50.62±4.13 and 44.30±12.66(%) (p<0.001)for each exposure group respectively after 24 h exposure. Detached germ cells counts significantly increased in each exposure group after 6,12,24 and 48 h exposure in a dose-and time-dependent manner (p<0.05 or p<0.001).2. After 24 h exposure, detached germ cells and adherent cultured Sertoli-germ cells were collected to detected the apoptosis status with annexin V binding assay. In each exposure group, early apoptotic cells proportion of detached germ cells significantly increased to 24.25±2.88, 33.08+2.43 and 29.23±2.54 (%) and late apoptotic/necrotic proportions of detached germ cells also statistically rose to 17.35±1.16, 23.14+1.06 and 20.70+0.48 (%), respectively (p<0.05 or p<0.001). FLC exposure obviously caused early apoptotic cells proportion in each exposure group to grow to 10.80±1.86,13.52±0.72 and 16.62±20.35 (%) (p <0.05 or p<0.001). However, there was no effect on Sertoli-germ cell late apoptotic/necrotic cell proportion.3. FLC down regulated caspase 8 gene expression in medium dose group after 24 h exposure (p=0.006). Down regulations of Bcl-2 gene expression were observed in each treatment group after 24,48 and 72 h exposure in a dose-and time-dependent manner (p<0.05 or p<0.001). FLC also raised Bax, Bad, Cytochrome c, caspase 9 and caspase 3 gene expression levels (p<0.05 or p<0.001). Up regulations of NF k B family members P50 and P65 gene expression appeared in medium and higher dose group after 72 h exposure (p<0.05 or p<0.001). p38 MAPK gene expression was up regulated after higher dose FLC exposed for 24 h (p=0.014), and each dose group exposure had this gene statistically up regulated after 48 and 72 h exposure in a dose-and time-dependent manner (p<0.05 or p<0.001).FLC affected primary cultured Sertoli cells.Testes of 18-21-old SD rats were aseptically removed and directed two steps of enzyme digestion which were used for the isolation Sertoli-germ cells. After 24 h of Sertoli-germ cell isolation, removal sperm cell by 20 mmol/L Tris-HCl to obtain purified Sertoli cells. Primary Sertoli cells in 2-5 days after purification were in the exponential growth phase and were chosen to carry on next experiment.1. MTT assay was carried on in control and each FLC treatment group after 6,12 and 24 h exposure. Primary cultured Sertoli cells viability obviously decreased in higher dose group after 12 h exposure (p<0.001). Each dose of FLC 24 h exposure reduced cell viability to 74.7±2.5,56.1 +4.4 and 52.3±6.4 (%) respectively (p<0.05 or p<0.001). And there was a dose- and time- dependent manner.2. Immunofluorenscence staining assay showed that FLC exposure for 6, 12,24,48 and 72 h changed the levels of TJs important component proteins which were Occludin and Z0-1. Medium and higher dose FLC exposure down regulated the Occludin protein expression after 6 h exposure (p≤0.001). There were significant down regulations in each dose groups after 24 and 48 h exposure (p<0.05 or p<0.001). Gene expression profiles indicated that higher dose FLC 24 h exposure leaded obvious down regulation in Occludin gene (p=0.006). Z0-1 protein of primary Sertoli cells in medium and higher dose FLC treated groups were statistically lower after 12 h exposure (p<0.001). This down regulation was enhanced after FLC treatments for 24,48 and 72 h (p<0.05 or p<0.001). ZO-1 gene expression was also depressed in a dose-dependent manner.3. FLC exposure for 6,12,2448 and 72 h changed Sertoli cell secretory function related genes expression. ABP genes expression was dose-dependent down regulated after FLC 24,48 and 72 h treatments. Tf expression performed a significant depression after 6,12 and 72 h higher dose FLC exposure (p<0.05 and p<0.001). Only higher dose FLC 72 h treatment caused INHB gene down regulation (p=0.031).Summarization of the above results indicated that FLC 28 days treated male SD rats experienced testes issue oxidative stress levels increase, serum T reduction, testicular seminiferous epithelium degeneration, Sertoli cell vacuolization, multinucleated giant cells containing engulfed bodies increase and cauda epididymal sperm counts negative changes. FLC caused apoptosis in primary co-cultured Sertoli-germ cell by inhibiting Bcl-2 expression and enhancing Bax and Bad genes expression. Intrinsic apoptotic signaling pathway might be one of the main mechanisms of apoptosis in this study. Simultaneously, NFκ B and p38 MAPK signaling pathways might also involve in regulation FLC induced primary co-cultured Sertoli-germ cells apoptosis via Bcl-2 expression depression and/or pro-apoptotic proteins Bax and Bad genes expression enhancement. In primary cultured Sertoli cells, FLC damaged BTB integrity by affecting TJs structural proteins including Occludin and ZO-1. At the same time, gene expressions of ABP,Tf and INHB proteins which reflect Sertoli cell secretory function were down regulated under the influence of FLC.Thus, it was proposed possible mechanisms of FLC testicular toxicity as following:a) FLC could change oxidative stress status in testes, cause testicular damages and disrupte the specific microenvironment during spematogensis;b) FLC could cause apoptosis in co-cultured Sertoli-germ cells mainly by intrinsic apoptotic signaling pathway with the assistant of NF κB and p38 MAPK signal pathway.c) TJs structural proteins in Sertoli cells could also be influenced by FLC directly. BTB integrity and Sertoli cell paracrine function were destroyed. High level T and iron essential for germ cell growth and development were no longer maintained.