Chronotoxicity Of Pesticide Fenvalerate With Mechanism Related To Male Reproductive Dysfunction

Fenvalerate(fenvalerate, Fen) is an important environmental chemical pollutants(ECPs) widely used due to its high toxicity, broad-spectrum and rapid degradation in the environment. Fenvalerate exposure can induce health effects including male reproductive dysfunction. In most organisms, physiological and behavioral functions change in terms of circadian rhythm. Because of this rhythm, the organisms show different sensitivity to the same drug at different times of a day, leading to different responses to the same dose of exposure but at different daily time. So far no circadian effects of fenvalerate on male reproductive functions are reported in humans.With joint methods of chronobiology and reproductive toxicology, this study is to explore the mechanism on chronotoxicity of fenvalerate, based on the findings that clock genes can regulate reproductive biological rhythm, in order to provide a novel way for the treatment and prevention of male reproductive dysfunction.Part 1 Circadian chronotoxicity of fenvalerate on reproductive functions in male ratsIn recent years, the adverse effects of fenvalerate exposure on reproductive system have attracted more and more attention at home and abroad. Researches show that fenvalerate can interfere with the endocrine function, bind to estrogen receptors to induce estrogenic activity, and mimic estrogen and antiandrogen activity in the reporter gene assay. It has been confirmed that fenvalerate can decline semen quality, affect the synthesis of testosterone to damage the male reproductive system.Objective This study is to explore the circadian chronotoxicity of fenvalerate on the male reproductive functions in rats to provide experimental data for the rational use of fenvalerate.Methods Rats were exposed to fenvalerate daily at different circadian times(CT9:00, CT13:00, CT17:00, CT21:00, CT1:00, CT5:00) for 30 days. Blood was collected from the ophthalmic venous plexus respectively on CT9:00, CT13:00, CT17:00, CT21:00, CT1:00 and CT5:00 and the hormones(Testosterone,estradiol,gonadotropin releasing hormone, luteinizing hormone, follicle stimulating hormone) in the blood were tested upon exposure to fenvalerate.Results Upon exposure to fenvalerate, the blood level of testosterone significantly decreased, while the levels of E2, Gn RH, LH and FSH increased. In the control group, the secretion of testosterone and estrogen showed circadian rhythms. The peak phases of testosterone at different circadian times were-136.57(CT1:00)、-170.06(CT5:00)、-34.92(CT9:00)、-85.08(CT13:00)、-89.13(CT17:00)and-120.29(CT21:00). The peak phases of E2 at different circadian times were-45.8(CT1:00)、-43.61(CT5:00)、-100.55(CT9:00)、-51.99(CT13:00)、-56.45(CT17:00)和-45.64(CT21:00). Exposured to fenvalerate, the blood level of testosterone decreased 5.7%(CT1:00), 10.4%(CT5:00), 16.7%(CT9:00), 13.1%(CT13:00), 20.6%(CT17:00), 10.4%(CT21:00)respectively,while estradiol increased 2.7%(CT1:00), 23.0%(CT5:00), 40.7%(CT9:00), 26.8%(CT13:00), 52.2%(CT17:00), 33.9%(CT21:00). The circadian rhythms of testosterone and E2 were altered after fenvalerate exposure, with the peak phases of testosterone at different circadian times of-127.78(CT1:00)、-155.42(CT5:00)、-191.9(CT9:00)、-183.06(CT13:00)、-170.78(CT17:00)and-147.97(CT21:00), and the peak phases of E2 at different circadian times of-45.8(CT1:00)、-43.61(CT5:00)、-100.55(CT9:00)、-51.99(CT13:00)、-56.45(CT17:00)和-45.64(CT21:00).The greatest changes in peak times between fenvalerate exposure and control group appeared at CT9:00. The peak time of testosterone in fenvalerate group was 10 h 27min later than that in control group. The peak time of E2 in fenvalerate group was 2h 29 min earlier than that in control group. The result indicated that animals treated at CT9:00 were more sensitive to the fenvalerate exposure than in other timepoints of a day.Part 2 The mechanism of fenvalerate chronotoxicity on male reproductive function in ratsThe circadian rhythm of mammal is controlled by biological oscillators via transcriptional- translational feedback loops of circadian genes and proteins. Mammalian clock genes include Per1/2/3, Cry1/2, Clock, Bmal1, etc. These clock genes have been found in the mammalian reproductive system one after another. Previous studies have confirmed that biological rhythm and clock genes are involved in reproductive function in mammals. The rate of sperm deformity was increased, and the secretion of testosterone was decreased in Bmal1 knockout mice.The disruption of Clock gene’s e xpression in the testis of male mice caused a significant decrease in the activity of sperm acrosin.Micro RNAs regulate the expression of their target proteins through the mechanism of post transcriptional regulation.Studies have indicated that micro RNAs participate in the regulation of circadian rhythm the synthesis of testosterone.Objective This study is to investigate the molecular mechanism of fenvalerate chronotoxicity on male reproductive function.Methods The rats exposed to fenvalerate at two daily times(CT9:00、CT21:00) were chosen to measure changes in expression of Bmal1 and the key enzymes for testosterone synthesis(St AR、HSD3b1、17β-HSD)in testis, and to screen differentially expressive genes by micro RNA microarray, with verification of the microarray results by PCR.Results The expression of Bmal1, St AR and 3β-HSD in the CT9:00 group were significantly down-regulated compared in the CT9:00 group with the control, while no obvious changes were found in the CT21:00 group. Changes in micro RNA expression were more apparent in CT9:00 group than in CT21:00 group. The expression patterns of mi R494-3p, mi R142-3p and mi R93-5p were chosen to be verified by PCR. The expression ofmi R494-3p, mi R142-3p and mi R93-5p were significantly up-regulated in the CT9:00 group compared with the control, while no obvious changes were found in the CT21:00 group. Mi R-93 regulated circadian rhythm through pre-transcriptional regulation, and Bmal1 was one of the target genes of mi R-494-3p and mi R142-3p.The results indicated that fenvalerate treatment could alter the expression of Bmal1 by micro RNA, to effect the level of the key enzymes in the synthesis pathway of testosterone, and finally to induce the chronotoxicity on the secretion of testosterone.Part 3 Joint effects of fenvalerate exposure and clock gene polymorphisms on male infertilityThe Clock gene is located on the long arm of chromosome 4q12. CLOCK and BMAL1 form heterodimer to regulate many clock genes and clock-controlled genes. Previous studies have confirmed that the Clock gene is also involved in the regulation of reproductive function. Infertility was noted in Clock gene knockout mice, with changes of hormone concentration.But, there is no relevant report in human reproduction.Male infertility, as a complex disease, is the result of the interaction of environmental and genetic factors. Fenvalerate is a new type II pyrethroid pesticide with potential health effects in exposed population, including male infertility.Objective This study is to explore the joint effects of fenvalerate exposure and Clock gene polymorphisms on male infertility.Methods A case-control study was conducted in 478 patients with idiopathic male infertility and 194 control patients, to analyze the association between the Clock tag SNPs(rs1801260,rs3817444 and rs3749474) and semen quality, to evaluate the influence of these genetic variants on levels of serum hormones. By using 3-(3-PBA, 3-pheno Xybenznicacid), the metabolite of fenvalerate as an internal exposure marker, a GMDR model was applied to evaluate the joint effects of fenvalerate exposure and Clock gene polymorphisms on male idiopathic infertility.Results Significant associations were revealed between genetic variant rs3749474 and sperm number per ejaculum, semen volume and sperm motility; between variants rs1801260 and sperm motility, and between rs3817444 and sperm motility. The concentration of 3-PBA in the urine of the patients was 1.41 ng/ml, which was significantly higher than that in the control group(0.75 ng/ml). The GMDR model analysis showed a joint effect of fenvalerate exposure with Clock gene polymorphisms( rs3817444 、rs1801260)on male idiopathic infertility, suggesting that the polymorphisms could be a potential risk factor of the productive dysfunctions induced by fenvalerate.Conclusions The chronotoxicity on male reproductive functions caused by fenvalerate treatment was revealed in rats, with CT9:00 as a sensitive daily timepoint upon exposure to fenvalerate. Circadian variation in micro RNA- Bmal1 gene- testosterone synthesis pathway was involved in the mechanism by which fenvaterate chronotoxicity was induced. In patients with fenralerate exposure and idiopathic male infertility, the SNPs of cloock gene was significantly correlated with impaired reproductive functions, and a joint effect of fenvalerate exposure and Clock gene polymorphisms on male idiopathic infertility was revealed, which may used as risk factor of reproductive dysfunction in fenvalerate exposed population.