| Objective:It is known that the organic compounds are about 7000,000 kinds in the world including 100,000 kinds' synthetic organic compounds. The numbers keep growing in the speed of more than 2,000 per year continualy. Many organic pollutants have certain of bioaccumulative or carcinogenic, teratogenic and mutagenic effects. Some of organic compounds can cause some irreversible effects on the reproductive function, which are invisible killer to human. At the present, many organic compounds were found in the source water and the tap water in many cities in China. Many organic compounds which are endocrine disrupter effect were found in the tap water in some cities. The problem about organic compounds is becoming more and more obvious. But our region has not yet carried out monitoring on environmental endocrine disrupters. The main source of drinking water is glacier melt water in Urumqi, so the organic pollutants may be different from that of the other reigons which source water were taken from the rivers. This study aimed to determine the main organic componds in source water and treated water in Urumqi city and test whether these organic pollutants has estrogenic activity. This study also aimed to explore the mechanisms of environmental estrogenic and explore possible water pollution biomarkers. Methods:1) In early August 2009,100L source water and treated water were collected from the three main water plants respectively in Urumqi. Each 20L water sample were extracted by a HLB solid phase extraction column. The organic componds is eluted from the HLB solid phase extraction column. After being dried up by the nitrogen, every 20L water sample was metered volume to 1ml by n-hexane. By GC/MS detecting, the mass spectras were obtained. According to the mass spectrums and the NIST2005 library researching, organic compounds were identified. The relative quanties of the organic compounds were caculated by the area of peaks of the organic compounds. The main organic compounds were determined in in water in Urumqi; 2) Retention time of every phthalate ester was determined by first-class mass spectrometer. After being explored the appropriate the collision energy of second-class mass the conditions of tandem mass spectrometry were optimized. The method of quantitative analysis of dimethyl phthalate, diethyl phthalate, n-butyl phthalate, isobutyl phthalate, amyl acetate phthalate esters by gas-triple quadrupole tandem mass spectrometry in the MRM mode was the established.5 phthalate esters were quantitative analyzed in the drinking water in Urumqi; 3) According to the quantitative results of phthalate esters and the peak area of the other substances, the concentration of various organic compounds in drinking water in Urumqi were obtained. According to the CAS number of organic compounds in water samples, LD50 data were searched in the"MSDS"and "Chemical book"database. At the same time, relevant national standards were searched, too. According to the LD50 of organic compounds which have national standards, the limits of the other organic compounds which have not the national standards were evaluated. Then, the limits of the organic compounds were introduced in the formula of Nemerow index. The score of Nemerow index is higher, the water quality is poorer; 4) 50 healthy ICR male mice were selected, which were weighted 15.93±2.67 grams. After 1 week adaptation fed, the mice were randomly divided into 5 groups.The concentrated extract organic compounds of treated water were dissolved in DMSO.5 dose groups were set, the control group (0.25 ml·kg-1DMSO group), low dose group (equivalent to 1.25L of original water·kg-1), medium dose group (equivalent to 2.5L of original water·kg-1), high dose group (equivalent to 5L of original water·kg-1),17β-estradiol (1mg·kg-1). The drinking water extracts were given to the mice daily by gavage. After 15 days, mice were killed and weighed. Eye blood of the mice were taken and the activity of SOD, MDA, ACP, AKP, y-GT of the testis and serum testosterone and follicle stimulating hormone were measured; 5) MCF-7 cells were cultured in 37℃,5% CO2,100% humidity in 96-well culture plates with phenol red-free DMEM/F12 medium containing hormone-free serum medium. MCF-7 cell with ethanol was solvent control group. MCF-7 cells with 17-β-estradiol were as the positive control group, which concentrations were 10-6mol/L,10-9mol/L, and 10-12 mol/L, respectively. The experimental groups were given by six water extracts, which were equivalent to the original water of 500ml,1000ml and 2000ml, respectively. Each dose has 4 duplicate holes. MCF-7 cell proliferation was detected by Alamar Blue respectively, in 12 hours,24 hours and 48 hours. Absorbance value and the proliferation rate were measured. MCF-7 cells were cultured in 12-well plates with phenol red-free DMEM/F12 medium containing hormone-free serum medium. According to the status of cell proliferation, two groups, middle dose group and high-dose group were set (that is, each group is equivalent to the original water of 1000 ml and 2000 ml. MCF-7 cells were cultured for 48 hours, and then exposed to the water extracts for 24 hours. Total RNA were extracted by trazol and reverse transcript. The samples were selected randomly and Hsp90AA1 gene, ERa gene, ERRa gene were amplified by ordinary PCR. The PCR products of these genes were showed by the gel electrophoresis. Under the UV light, the products of these genes were cut and then pured. Each pure gene product, respectively, were diluted with dEPC water as the concentration gradient of 101,102,103,104,105,106,107, and dEPC water was as Negative control. The standard curve of each gene were prepared by real-time fluorescence quantitative PCR. Hsp90AA1 gene, ERa gene, ERRa gene expression quantitaty were detected; 6) The MCF-7 cells were cultured in 24-well culture plates with phenol red-free DMEM/F12 medium containing hormone-free serum medium.17-β-estradiol (E2) (10-9mol/L) was geiven to the cell as a positive control and the experimental groups were six water extracts. Creep plates of the highest dose group were collected. The immunochemical detection of fluorescence measured Bax protein and Bcl-2 protein expression, Bcl-2/Bax ratio was calculated. Results:1) The main organic componds in source water and treated water in Urumqi city were 8 large classes of 39 species, including alkanes, esters, carboxylic acids, aromatics, ketones, alcohols, olefin and amide. Hydrocarbons were detected in all water samples which were the largest organic componds in the total, and followed by esters, carboxylic acids, aromatic compounds. Compared to the similar studies in other provinces, the types of pollutants of surface water in Urumqi City are less; 2) There are six phthalate esters in the source water and treated water of Urumqi city, which were listed in the EPA blacklist. Six phthalate esters are dimethyl phthalate, dibutyl phthalate ethyl ester, dibutyl phthalate, iso octyl phthalate esters, butyl octyl phthalate esters, dibutyl phthalate (2-methyl propyl) ester; 3) The removal rate of hydrocarbons, esters, carboxylic acids in A plant is higher than that in the B Plant and C plant; 4) The Nemerow score of source water in A plant is much higher than the other water plants, which suggesting that organic pollution in source water in A plant is seriously than in the other water samples. The Nemerow score of source water were higher than the treated water in every plant. The treated water quality is better than that of the source water; 5) The organic componds in three water plants in Urumqi has not showed the clear anti-androgen effect on male mice; 6) After 24 hours'exposure, MCF-7 cells proliferation of high dose and middle dose groups was significantly different from the negative control group.There were no significant difference between the low dose groups and the negative control groups. The middle dose positive group and each water samples groups have the significant difference. After 24 hours'exposure, MCF-7 cell proliferation rate of the middle dose positive control was the highest and the high dose group has the stronger effect in promoting proliferation than the low-dose groups. But after 48 hours' exposure, almost all of the low dose groups showed the greater proliferation rate than the the high dose group.7) There are significant differences between the gray value of Bax protein fluorescence in each group.The gray value of Bax protein fluorescence in the experimental groups were lower than that in negative group.Except C plant treated water group, the fluorescence gray value of the other Bcl-2 protein in each experimental group were higher than the negative group. Bcl-2/Bax ratio in each group were significantly different (P<0.05). Bcl-2/Bax ratio in each experimental group was higher than the negative group (P<0.05); Bcl-2/Bax ratio of A source water group, B source water group and C 7source water group were higher than group A treated water, B and C treated water group treated water group, respectively (P<0.05); 8) In the high dose groups, there are significantly different between the hsp90AAl expression of the source water and the treated water. MCF-7 cells hsp90AA1 expression in high dose group of source water in B plant was upregulated and was significantly higher than A, C water plant, but MCF-7 cells hsp90AA1 expression in high dose group of treated water in B plant did not show significant differences compared to the other water plant; Hsp90AA1, ERa, ERRa gene mRNA expression was significantly correlated and the Pearson correlation coefficients were 0.988 and 0.944, respectively (P<0.05). Conclusion:The organic pollutants in drinking water in Urumqi are less than the other aera in China. The MCF-7 cell proliferation phenomenon caused by water extracts may not be typical of the nuclear receptor pathway through the transcription of ERa, which is more dependent on non-genomic effects of MCF-7 cell proliferation. Hsp90AA1 expression of MCF-7 was sensitive to the environmental pollutants and there are the relevance among the hsp90, ER and ERRa, so we recommend hsp90AA1 as markers to measure the effects of environmental estrogens. |
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