| Objective: Recently, environmental endocrine disrupting chemicals (EDCs) have been abundantly found in the environment. Their potential harms to the human health and wildlife have become the focus of world-wide researching, in particular, some developed countries such as America, Germany, and Japan have paid much more attention to their hazards. In 1995, World Health Organization (WHO) declared that phthalic acid esters (PAEs) could disrupt the functions of the endocrine system and their hazards must be controlled. Now, it has been suggested that PAEs is one of the global environmental pollutants. Some of them, such as di-n-butyl phthalate (DBP) and di-(2-ethylhexyl) phthalate (DEHP) have been described as being among the most ubiquitous man-made environmental contaminations. DBP and DEHP have been classified as U.S. EPA's priority chemical pollutants for controlling. To evaluate the toxicity of PAEs on the reproduction-endocrine system and the potential hazards to the human health, DBP and DEHP were taken as the tested chemicals of this study, the estrogenic activities of DBP and DEHP and their effects on the ovarian functions of adult female mice were examined. This study made up the inadequation on toxic effect data of DBP and DEHP on the ovarian functions of adult female mice and provided scientific basis for overall evaluating the toxicological effects of PAEs.Methods: (1) In vitro assay: Human estrogen-dependent MCF-7 breast cancer cells were cultured in RPMI1640 medium containing 10% fetal bovine serum (FBS). Five days before the addition of the tested compounds, the cells were washed by phosphate balanced solution (PBS), and the medium was substituted with a phenol red-free RPMI1640 medium containing 5% charcoal dextral-stripped FBS. The respective test compound was added into fresh medium and the negative control cells received only the vehicle (0.1% ethanol). In the treated groups, the cells were treated with 10'10mol/L~10"3mol/L DBP and 10"8mol/L~10"3mol/L DEHP, respectively. In the postive control groups, the cells were received 10"9mol/L 17 ^ -estradiol (E2). The proliferation of MCF-7 cells was analyzed by the MTT assay, growth curves, mitotic index, plating efficiency and coloning efficiency. (2) In vivo assay: The healthy Kunming adult female mice with body weight from 22g to 30g were randomly divided into DBP and DEHP groups and control group, 16 female mice per group. There are three exposure groups and one reagent control group for each tested compound. In the DBP exposure groups, the mice were administrated by peritoneal injection with DBP at the doses of 400 > 40 and 4mg/kg body weight, respectively. In the DEHP exposure groups, the mice were administrated by the same way with DEHP at the doses of 1250^ 125 and 12.5mg/kg body weight, respectively. The mice in control groups were administrated with the same amount of vehicle (dimethyl sulfoxide, DMSO). All the mice were administrated once a day, continuously for 14 days. The estrous cycles were observed during the period of the whole experimental study in vivo. On the fifteenth and thirtieth days, the mice were sacrificed. The organ coefficients of uterus and ovary were determinded, and the histopathological changes of the left ovary were observed.Results: (1) In vitro assay: Compared with the ethanol control cells, the proliferation of the tested cells treated with 10"10~10"3mol/L DBP and 10"5~10 3mol/L DEHP, like E2, was markedly enhanced, and the activity of the cell proliferation reached the maximum at 10'5mol/L DBP and 10"3mol/L DEHP, respectively. The relative proliferation effects of 10"5mol/L DBP and 10'3mol/L DEHP were 98.16% and 90.92% in comparison with 10"9mol/L E2, respectively. The relative proliferation potency of DBP and DEHP were 10"4 and 10"6 in comparsion with E2, respectively. During log phase, the mitotic index of the tested cells treated with DBP and DEHP, as well as E2, was significantly increased.
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