| At present, with the development of knowledge in the area of the health damage induced by endocrine disrupting chemicals (EDCs), standards for allowable levels have been set by international organizations, eg.50μg/kg.d for bisphenol A (BPA) and ethinyloestradiol (EE2),5μg/kg.d for nonyl-phenol (NP). However, more and more people still concern about the potential health hazard of EDCs. We here carried out a related research to further explore whether BPA induce health hazard under the condition of low-dose and long-period exposure.Part I Contents and distribution of EDCs in foods available in marketObjective:To set up an integrated method used for pre-treating, extracting, refining and detecting on EDCs in foods. Based on it, concentrations of eight selected EDCs in nature foods purchased from markets were tested simultaneously, which will be the important foundation of the further risk assessment on EDCs exposure in diet.Methods:According to the geographical characteristics of the river basins in China, a cross-sectional investigation at three Chinese cities, i.e., Wuhan (WH), Guangzhou (GZ) and Jinan (JN) were include in the study.450food samples representing7food categories, collected from three geographic cities in China, were analyzed for eight closely watched EDCs using high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). The effect of co-exposure to multiple EDCs was measured by the total estrogen equivalent, which was thought to be better for representing the level of co-exposure.Results:(1)8selected EDCs in food samples can be effectively separated by liquid phase technique and detected by mass spectrometry method when we pretreated and detected the samples by the optimized parameters. A good linear relationship between the content and the area of chromatographic peak was observed in the standard series of EDCs components.(2) Except for estrone (E1),7out of the8selected EDCs were detected in various foods and differentiation in positive rate was found between food categories. For the same order, the top three detected rate of EDCs component were NP>BPA>EE2.(3) Spearman correlation analysis revealed that the association between NP and OP, EE2and OP, BPA and OP, EE2and NP, BPA and NP, BPA and EE2, E2and E3presented lowly positive correlation (P<0.05).(4) The difference between the levels of E2and total estrogen equivalent (∑EEQ) was calculated by Wilcoxon method and the result showed a statistically significant difference (P<0.05).(5) Correspondence analysis was used for observing the orientation of different food categories and EDCs, the results showed inclinations of various EDCs always occurring in special food category.Conclusion:In present study, we established a set of methods for detecting multiple EDCs by LC-MS/MS simultaneously. Based on the methods, we carried out a cross-section investigation on the EDCs in various foods purchased from3cities which seated in different river basins. The results showed low levels of EDCs isomers in foods and inclinations of various EDCs always occurring in special food category in all sampling cities. However, the co-exposure could magnify the health hazard of EDCs.Part II Probabilistic assessment on dietary exposure to EDCs Objective:Based on the dataset of EDCs levels combined with the food consumption of Chinese, a probabilistic simulation of EDCs exposure was conducted by using the Monte Carlo method, which provided a basic evidence for the further risk analysis and management for EDCs.Methods: According to the professional knowledge, a mathematic model for calculating the EDCs exposure was established firstly and the parameters of variables (EDCs level, body weight, food consumption data, etc.) from the model were simulated by using Crystal Ball11.0Software. The simulated parameters for describing the distributions of variables were inputted the program when we calculate the individual exposure. If we calculated a large number of individual values, we could analyze the frequency distribution of individual exposure and the probability.Results:After calculating a large number of individual exposures and analyzing the frequency distribution, we found the population from3cities was identical underwent a low dose of EDCs exposure such as0.27μg/kg.d in GZ,0.28μg/kg.d in WH,0.28μg/kg.d in JN for BPA.Conclusion:Chinese population underwent a low dose of EDCs exposure based on the existing EDCs levels in foods and their dietary custom. However, considering of the co-exposure and various exposure pathways, further research was still necessary.Part III Reproductive toxicity in male rats induce by low-dose BPAObjective: Established BPA treated male rat model characteristic of long-term and low-dose BPA exposure and based on the model, series of detection were conducted to observe whether BPA could damage spatial memory of rats and explore its reproductive toxicity.Methods:After2weeks adaptive feed,30Wistar rats aged30days were randomly divided into3groups, control, lOμg/kg.d and50μg/kg.d BPA groups. The rats of control group were fed with corn oil solvent and the food-intake and weight of rats from each group were recorded. After expose to BPA for8weeks, we executed male rats mate with non-exposed females in estrus. After1week from the mating point, all FO rats were introduced to the Morris Water Maze (MWM) test for one week then were sacrificed for testing their serum free BPA levels, coefficients of testis and epididymis, deformity rate of sperm, pathologic morphology of testicular tissue, et.al. What is more, the apoptosis of testicular tissue was detected by using TUNEL assay and the expression of PCNA and acetyl-protein were detected by using immunohistochemical assay. Their offspring (F1) received no treatment until behavioral testing at the age of PND56for the MWM by using the same methods as their fathers. Food intake and body weight of all F1animals were routinely recorded.Results:(1) There was no significant difference between control and BPA male rats in daily food consumption and body weight. However, the serum level of free BPA was differing from one another between groups.(2)50μg/kg.d BPA rats from FO swam longer distance (P<0.05) and spent more time with faster swimming velocity (P<0.05) to find the hidden platform than did the control counterparts, they also made shorter duration in the platform quadrant (P<0.05) relative to the controls. Except for duration in the platform quadrant, F1offspring from BPA fathers were identical to their father in distance, velocity and latency (P<0.05).(3) There were no significant differences between control and BPA male rats in coefficients of testis and epididymis, deformity rate of sperm.(4) There was no significant difference between control and BPA male rats in apoptosis of testicular tissue. However, reduced layer of cell and expression of PCNA in seminiferous tubule were observed in50μg/kg.d BPA rats (P<0.05).(5) Results of Western blotting showed the expression of acetyl-protein decreased at25KD in testicular tissue of rats underwent50μg/kg.d BPA.Conclusion:Male rats exposed to50μg/kg.d BPA for long-term could damage their spatial memory ability and pass on to the next generation, and the decreased expression of acetyl-protein in testis may be partly responsible for the effect. |
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