Study On The Male Reproductive Toxicity Induced By Organic Extracts From Jialing River In Chongqing City

Water pollution by organic micropollutants is one of the most critical problems concerning drinking water resources and environmental protection of water bodies.Surface waters are contaminated by numerous organic pollutants including pesticides, polychlorinated biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs).These substances enter freshwater ecosystems from agricultural areas, urban areas, and industrial production sites by drainage, superficial runoff, rainwater, as well as discharge of wastewater.China's extraordinary economic growth, industrialization, and urbanization, coupled with inadequate investment in basic water supply and treatment infrastructure, have resulted in widespread water pollution. By the year 2000, the volume of wastewater produced could double from 1990 levels to almost 78 billion tons. These are alarming trends with potentially serious consequences for human health. Chinese cities have limited facilities or infrastructure to treat sewage or drinking water, which mostly comes from surface water of large rivers or lakes. The Jialing River are a vital source of drinking water for a large number of Chongqing people, we have detected the organic pollutants from the Jialing River water.Humans are exposed to mixtures of chemicals, rather than to individual chemicals. From a public health point of view, it is most relevant to answer the question of whether or not the components in a mixture interact in a way that results in an increase in their overall effect compared with the sum of the effects of the individual components. The combined effects of such complex pollutant mixtures on human health are widely unknown. They may act additively, synergistically, or antagonistically. The best way of approaching the problem of risk assessment is to use biological tests that give a global response to the pool of pollutants present. If biological tests can be accomplished with the chemical characterization of toxic pollutants, the effectiveness of measures for pollution reduction will be improved. Spermatogenesis is a dynamic and highly synchronized process that takes place in the seminiferous tubules of the testis. This process begins with the clonal expansion of stem spermatogonia and involves mitosis, meiosis, and cellular differentiation. As in many tissues throughout the body, the number of cells in the seminiferous tubules of the testis is determined by a dynamic balance between cell proliferation and apoptotic cell death. various types of testicular injuries, including hormonal perturbations , heat exposure , Sertoli cell toxicants such as 2,5-hexanedione and mono-(2-ethylhexyl) phthalate, and germ cell toxicants like x-irradiation, all result in germ cell apoptosis. Together, these observations indicate that the seminiferous epithelium responds to most adverse environmental conditions by eliminating germ cells through programmed cell death. And since Sertoli cells are responsible for establishing the environment within the seminiferous epithelium, this implies that Sertoli cells have a way to initiate and control germ cell apoptosis. Germ cell apoptosis can be triggered by a deficiency of a pro-survival factor, by an excess of a pro-apoptotic factor, or by synergistic alterations in both pathways. We know about the Scf/c-kit and FasL/Fas systems as the most important pro-survival factor and pro-apoptosis.The aim of the present study was to evaluate the male reproductive toxicity of the organic extracts (OE) from Jialing River. Our research was divided into three parts:Part1: Separated organic extracts from the river water and identified the organic compounds in the river waterPart2: Evaluated the male reproductive toxicity with OECD 421 testPart3: Explored the molecular mechanism of testis toxicity induced by organic extractsThe main results are as follows:The trace organic pollutants of river water in Chongqing city were concentrated on XAD resins,then the organic pollutants was analyzed with GC/MS.It indicated that there were 8 organic pollutants in water samples,including 6 PAHs and 2 PAEs. Di-n-octyl phthalate and Fluoranthene are listed as top pollutants of environment in China.Organic pollutants were extracted with XAD-2 resin from river water and then administered to male and female Sprague-Dawley rat by gavage in different dosage of 2,16 and 80 L/kg.bw/day for 28d. On the 28th day after treatment,the male rats were killed and the testis,epididymis were removed and fixed for pathohistological examination.The growth and general appearance of the treated animals were normal throughout the experiment. Final body weights in the treated and control groups were not significantly different between different groups. Vacuoles Degeneration of seminiferous epithelium was observed at the treated group, hyperplasia of Leydig cells only in the rat treated at the dosage of 2L/kg.bw/day, the atrophy of leydig cell could be observed at the 16L/kg.bw/day and 80L/kg.bw/day group. The weight of the epididymis was increased significantly at the dosage of 80L/kg.bw/day (P<0.05). The seminiferous tubules shrunk at the 16L/kg.bw/day and 80L/kg.bw/day group.The results suggest that the organic pollutants in river water is slightly harmful to male reproductive system,with the testis as target organ.Sperm counts of caudal epididymis were also evaluated. Caudal sperm counts showed a dose-dependent decline in all the groups. The rates of the sperm counts/epididymis weight were significant increased at 80L/kg.bw/day group compared to the control group. Percentage of abnormal sperm was determined. There were no statistically significant differences in whole abnormalities among groups. The percentages of sperm with abnormal head increased significantly in the 80L/kg.bw/day treatment group.The fertility of the male mice was assessed by the incidence of pregnancy in females.Pregnancy rate was 90% in control female group (9/10) and 70% in treated females group mated with treated males (7/10). Other reproductive parameters determined were the number of litters, implants, dead fetuses, and fetal body weight. No significant differences were found in fetal data of the control females mated with the treated males as compared to control group.Serum hormone level, including Testosterone, FSH, LH and E2, were measured using radioimmunoassay. Exposure of rats to organic extracts resulted in a significant decrease in testosterone levels at high dosage. Serum FSH level increased at at the 16L/kg.bw/day and 80L/kg.bw/day group compare to the control group.The TUNEL assay for in situ detection of apoptosis was performed in testis cross sections. In the control and treated group rats, a few TUNEL-positive germ cells were found. The cell types undergoing apoptosis tubules were spermatogonia and spermatocytes.There was no statistic difference between the control group and treated group .In contrast, organic extracts exposure resulted in a marked increase in the incidence of germ cell apoptosis, The apoptotic cells were identified as spermatids, which were the prominent cells undergoing apoptosis after exposure.Using immunohistochemistry and western blot analysis, we examined the changes of scf, fas ,fasL and c-kit protein in the rat testis. The percentage (%) of apoptotic spermatid cells significantly increased at 80L/kg.bw/day group compared to the control groups. Immunohistochemistry revealed that spermatocytes overexpressed fas, fasL and c-kit at 80L/kg.bw/day group. Western blot analysis demonstrated a dosage dependent increase in the production of Fas, FasL and c-kit after organic extract exposure. The findings of our study suggest that the organic pollutants of river water result in spermatid apoptosis and spermatogenesis arrest.In conclusion, organic extracts from the Jialing River water can induce testicular toxicity in our research, and sperm cell may be the target point. Organic pollution in river water is a potential threaten to the human reproductive health. We can not find the clear reproductive injury of F1 rat, and further work should be done. The information on the relationship between water organic pollution and human reproductive health are needed for the human reproductive health safe, and we should look for the sensitive biomarker to diagnosis the damage at the early stage of exposure.