| Objective:Environmental Endocrine-Disrupting Chemicals (EDCs) are a category of compounds that have the potential ability to interfere with the normal functions of the endocrine system, thereby leading to dysfunction of other organs in experimental models, wildlife and human beings. Fenvalerate (Fen), a type II pyrethroid, has been widely applied in agricultural settings and homes since 1970s. It was cited as a moderately hazardous pesticide by world health organization (WHO) in 1990. Recently, studies suggest that Fen has some unclear adverse effects on multiple organ systems including the reproductive system. The current studies proposed in this dissertation sought to explore the influence of Fen on uterine fibroids and spermatogenic function, and potential mechanisms of its effects. Our results will provide new evidence to further understand the mechanism(s) of reproductive toxicity of Fen, and contribute to the optimal management of exposure of certain populations to this EDC.Materials and Methods:For the female effects, human uterine leiomyoma cells (UtLM cells) and human uterine smooth muscle cells (UtSMCs) were used as in vitro models in this study. We first evaluated the influence of Fen on uterine fibroids by measuring proliferative responses and production of extracellular matrix (ECM) in UtLM cells and UtSMCs. Expression of p27 was determined by RT-PCR and Western blot analysis. By using an adenovirus p27 Tet-off system, we established cells that overexpressed p27; in contrast, "knock-down" expression of p27 was achieved by specific siRNA targeting of p27. Therefore, we explored whether the proliferative responses to Fen in the two cell lines would be altered in above-mentioned scenarios. For the male effects, male ICR mice were employed as an in vivo model to evaluate Fen exposure on male reproductive functions, and expression of calmodulin (CaM). Various methods were tested to obtain spermatocytes for further experiments. Finally, expression of CaM and intracellular calcium concentrations were determined in GC-2 cells with or without Fen.Results:(1) After treatment with 10 μM or 100 μM Fen for 24 hours, cell proliferative indices were increased in UtLM cells by a MTS assay. Meanwhile, similar trends were detected in UtSMCs. Furthermore, DNA synthesis was determined by a BrdU incorporation kit. Our results indicated that both UtLM cells and UtSMCs incubated with Fen showed exaggerated DNA synthesis, compared to cells in control medium. The proportion of UtLM cells and UtSMC in S phase was significantly increased by adding 10 μM Fen in the culture medium for 24 hours, as determined by cell cycle analysis. In contrast, the fraction of apoptotic cells was reduced in both cell types stimulated by Fen as determined by flow cytometry.(2) By real-time PCR, mRNA levels of type I collagen were determined to increase in a time-dependent manner due to Fen in UtLM cells and UtSMCs, respectively. Similarly, collagen protein in cell lysate and supernatant was up-regulated by Fen stimulation in a dose-dependent manner in UtLM cells and UtSMCs, as indicated by western blot analysis.(3) We established an estrogen-receptor reporter gene system by transfecting estrogen receptor plasmid coding additional luciferase reporter gene into UtLM cells and UtSMCs, respectively. As a positive control, we found E2 at the concentration of 1.0 nM triggered 6.14-fold increase in luciferase activity, which was substantially inhibited by ICI 182,780 at the dose of 1.0 μM. However, Fen at concentrations of 0.1 μM,1.0 μM or 10 μM did not significantly alter luciferase activity. Furthermore, we used a Receptor Competitive Binding Assay kit by fluorescence polarization to measure the binding affinity of Fen to ERoc or ERβ. Our data indicated that binding affinity of Fen to ERs was non-detectable at a dose range from 1 nM to 262.1 μM. Collectively, our results suggest that estrogen receptors less likely mediate the above effects by Fen.(4) Levels of mRNA expression of p27 were found to decrease in a time-dependent manner in UtLM cells and UtSMCs treated with Fen. With confocal microscopy, p27 was observed to be mainly expressed in the nuclear compartment, while 10 μM Fen resulted in a considerable decline in intensity of positive staining signal for p27.(5) An adeno-p27 Tet-off system was employed to overexpress p27. By western blot analysis, we found that p27 expression started to increase 4 hours after transfection. Cell counts showed increases of 126.97% and 133.33% in UtLM cells and UtSMCs, respectively, after 24 hours of Fen treatment. However, in the presence of overexpressed p27, this proliferative effect was abrogated. By contrast,1 μg/mL doxycycline (Dox) (that turns off p27 expression) restored the proliferative response induced by Fen to the extent of 125% and 132.43% in UtLM cells and UtSMCs, respectively. Furthermore, CDK2 mRNA was increased by 170.28% in UtLM cells and 170% in UtSMC cells, and this increase was eliminated by transfection of the p27 plasmid, while additional Dox rebounded changes in CDK2 mRNA to 166.81% and 189.25% in the UtLM cells and UtSMCs, respectively.(6) By real-time PCR, we discovered that chemically synthesized anti-p27 siRNA was able to knockdown mRNA expression of p27 to 28.62% and 34.36% in UtLM cells and UtSMCs, respectively, with accompanying decreases in the proliferative indices. Moreover, the siRNA could further decrease p27 expression even in the presence of 10 μM Fen to 48.7% and 46.5%, with accompanying enhanced proliferative indices of 126.42% and 125.52% in UtLM cells and UtSMC, respectively.(7) In assessing the male effects of Fen, the LD50 of Fen in ICR male adult mice was determined to be 185.62 mg/kg/bw by the Bliss method. After that, a dose response experiment was executed to explore the reproductive toxicity of Fen. By measuring the ratio of testis/body weight, exposure to Fen at a dose of 37.12 mg/kg/bw was found to result in a significant decrease in the ratio of testicular weights to body weights, accompanied by reduction in sperm motility and an increase in the percentage of head abnormalities. By H&E staining, we detected a reduction in layers and increased numbers of focal spermatocytes in testis from Fen-treated mice. When exposure dosages were increased, the pathogenic changes were exacerbated.(8) By immunochemistry staining, calmodulin was determined to be expressed in testicular tissue, mainly in the pachytene spermatocytes. Moreover, enhanced positive signals were detected in mice exposed to Fen for 4 weeks. By real-time PCR, calmodulin mRNA expression was found to increase in testicular tissues from mice treated with Fen, in a dose-dependent manner. This trend was further confirmed by western blot analysis.(9) A combination of collagenase and DNase I was added to minced testicular tissues, and incubated for 30 mins. Vitality of 97% was determined in final cell pools by Trypan Blue staining. Moreover, a discontinued percoll method was utilized to further purify cell fractions. Under microscopy, we observed that the majority of the cultured cells were degraded after 48 hours, although they could maintain normal morphology for the first 3 hours following isolation. Our findings suggest that acute isolated spermatocytes are not suitable for further experiments.(10) In order to explore the role of calcium signaling in Fen’s toxicity to spermatocytes, GC-2 cells were selected for our in vitro model. Based on our pilot study,5 μM was selected as a working concentration for further experiments. Protein expression of calmodulin was detectable in normal GC-2 cells as determined by immunostaining and western blot analysis, and exaggerated by treatment of Fen. By real-time PCR, mRNA expression of CaM increased after 6 hours incubation with Fen, and peaked at 24 hours.(11) We observed an increase in intracellular calcium concentration due to 5μM Fen under confocal microscopy, by labeling calcium with Fluo-3,AM. By replacing extracellular fluid with calcium-free buffer, we were still able to detect the enhanced intracellular calcium signals due to Fen. Thereby,2μM thapsigargin (TG) was used to pretreat GC-2 cells for 30 minutes to empty calcium in endoplasmic reticulum. After that, Fen was still shown to boost intracellular calcium concentration ([Ca2+]i). Collectively, these results suggest that both the extracellular influx of calcium and release of calcium pools in the endoplasmic reticulum participant in Fen-induced enhanced intracellular calcium.Conclusions:1. Among a certain range of doses, Fen induces cell proliferation and increases ECM synthesis in UtLM cells and UtSMCs, suggesting its role as a potential environmental risk factor for uterine fibroids; p27 mediates above effects induced by Fen.2. Fen results in dysfunction of the reproductive system in male mice, accompanying upregulation of calmodulin in the testis; Fen increases expression of calmodulin and concentration of intracellular free calcium in GC-2 cells. |
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