| Organochlorine pesticides(OCPs) such as DDT and BHC had ever used extensively and massively in worldwide. They have been banned to produce and use for more than twenty years, however, because of their hard degradation, high residue, iposolubility, and their biological amplification through food chain, which had made great harms to environmental ecosystem, animals, plants and human being. A great number of studies have indicated that DDT, BHC and their metabolites can still be detected in various environmental and biological samples. Their detected percents and concentrations were high in certain environments and population, especially in fatty tissue. Recent studies suggested that OCPs, as a type of persistent organic pollutants (POPs), possess endocrine dirupting effects. They may be concerned with decrease of sperm count and vitality, and increase of incidences of male infertility, carcinoma of testis and prostate. They may also be related to increase of incidences of prematuration, endometriosis, carcinoma of uterus, ovary and mammary gland. For these reasons, hazards of OCPs to human, especially to genital system, have caused great attention, and their reproductive and developmental toxicity have become a focus in recent studies. We selected the people as our studied objects who came from Tianmen city in Hubei province, because their average concentration of DDT residues in the fat samples of female residents was the highest in China in 1985. In the present study we determined residual levels of OCPs in sera of the local population, concentrations of FSH,LH,E2 and P in blood, and expressional abundance of the genes and proteins such as α-ER,β-EP and GnRH in the samples. Population survey and animal experiment were integrated to explore endocrine dirupting effects of OCPs.Part â… Effects of p,p'-DDE and beta-BHC on reproductionand development in pregnant mice and their foetus Objective: To explore effects of reproduction and development in pregnant mice and their foetus co-incubated by p,p'-DDE and beta-BHC.Methods: The pregnant mice were randomly divided into four groups, which were provided with plant oil, 1 mg/kg + 1 mg/kg, 5 mg/kg + 5 mg/kg, 10 mg/kg +10 mg/kg of p,p'-DDE and beta-BHC, respectively, from the 12th day to 14th after pregnancy and were treated one times per day. From P0 to P18, the pregnant mice were observed and weightened per day. When the animals were killed (P18), their blood was collected to determine concentration of estradiol and progestone by Immunoenzymetric assay (magnetic solid phase), and their placentae were collected to detect expressional abundance of α-estrogen receptor(α-ER), β-endorphin (β-EP) and gonadotropin releasing hormone (GnRH) mRNA by RT-PCR. The organ coefficients of heart, liver, kidney, spleen, pituitary, ovary, uterus, intraluminal fluid and placenta were determined, respectively. Corepus luteums, nidations, live foetus, dead foetus, absorbed foetus and featus deformities were counted, respectively. Weight, body length, tail length, abdominal circumference, anogenital distance (AD) and ratio of female to male (F/M) in the foetus were determined, respectively. The Spearman method was employed to analyse correlativity between expressional abundances of the genes and concentrations of the hormones, between AD and ratio of F/M and genes and hormones, respectively.Results: a) Effects on the pregnant mice: The pregnant mice were poisoned by p,p'-DDE and beta-BHC, which have decreased their weights and gained weights, increased organ coefficient of liver, uterus and intramminal fluid, decreased nidation, increased concentrations of estradiol and progestone in sera (P<0.05). b) Effcets on the foetus: with increase of p,p'-DDE and beta-BHC, the number of live foetus lessened while dead foetus, absorbed foetus and featus deformities increased, AD and ratio of F/M in F1 generation increased. Significances were found among the control, low-, mid-, high-dose groups. Expressional abundances α-ER and GnRH mRNA increased while β-EP decreased in placenta. There were significant differences among the four groups (P<0.05). c) Correlation analysis indicated that positive correlatons were found between concentrations of estradiol, progestone in sera and expressional abundances of α-ER, GnRH in placenta, between AD of foetus, ratio of F/M and α-ER, GnRH, between progestone and ratio of F/M, that negative correlation existed between estradiol, progestone and β-EP mRNA, between AD of foetus, ratio of F/M and β-EP mRNA. Conclusions: p,p'-DDE and beta-BHC possess estragenicity in female, which may disrupt reproduction and development, thereby lead to feminization in their offsprings.Part â…¡ Determination of residues of DDT and BHCin sera of pregnant women at deliveryObjective: To detect concentrations of the eight isomers of DDT and BHC in sera of pregnant women so as to reveal current residual levels of OCPs in population. Methods: Sera of the 71 women who delivered their babies at term in Tianmen hospital for maternal and child care in Hubei province during Jan 1st to Apr 30 in 2004 were collected. Capillary gas chromatography (CGC) was employed to determine concentrations of residues of DDT and BHC in sera.Results: a) The average recoveries of the three spiked levels were 87.3%～94.6%. The coefficient of variation (CV) were 1.8%～6.8%. The minimum detection limit was 0.0005 μg/L. b) The detected percent and range of detection of the eight metabolites were 7.04%～47.89% and 0.0～147.7 μg/L, respectively. The average concentration of γ-BHC among the BHCs and o,p'-DDT among the DDTs were the highest in the samples, respectively. c) The average detected percent and concentration of T-OCPs were 87.32% and 37.48±51.24 μg/L in sera of the 71 women, respectively. 9 of whom were taken as control because no metabolite was found, 26, 17, 19 of whom were divided into the low-, mid-, and high-residue groups because their average levels of T-OCPs were failed into 2.1～9.7 μg/L, 12.6～39.6 μg/L, and 54.5～248.1 μg/L, respectively.Conclusions: High residual levels of DDT and BHC are still detected in some people in China. Some products such as lindane and dicofol are probably used illegally in certain areas. It is very necessary to continue to determine residues of OCPs in environment and population.Part â…¢ Epidemiologic survey on the sampled populationObjective: To explore reproductive and developmental toxicity of OCPs, to rule out confounding factors effecting its endocrine disrupting effects, thereby increase reliability of the gained data.Methods: According to 《questionnaire on pregnant women's health》 , the sampled women were surveyed by the trained investigator in two days after they delivered their babys. Results: a) The responsivity to questionnaire was 100% (71/71). b) With increase of residues of OCPs in the pregnant women, the mean of previous adverse pregnancy outcomes (PAPO) went up (P < 0.05) . c) The change of mean of weight in neonates were probably related to residues of OCPs in the pregnant women. d) No significance was found on age, occupation, expose to harmful physical and chemical factors, drug use during pregnancy, smoking and drinking, nutrition and sleep quality during pregnancy, family medical history (FMH) and so on in the sampled women and their spouses (P>0.05).Conclusions: Epidemiological survey on population increases reliability of the other experimental data. Meanwhile, it indicates that residues of OCPs in body are probably concerned with reproduction dysfunction and maldevelopment.Part IV Effects of residues of OCPs on the four reproductive hormones in pregnant womenObjective: To explore effects of residues of OCPs in pregnant women on concentrations of follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2) and progesterone (P) in the sera of venous blood and blood from umbilical cord.Methods: The 71 pregnant women who delivered at term in Hubei Tianmen Hospital for Maternal and Child Care during Jan 1st to Apr 30th in 2004 were recruited to participate in the present study. 10 ml venous blood samples respectively from the studied individuals and umbilical cords at delivery were collected and analyzed by adopting Enzyme Immunoassay (Magnetic Solid Phase) to determine concentrations of FSH, LH, E2 and P. Spearman method was employed to analyze correlation between concentrations of T-OCPs and the four hormones, between levels of FSH, LH, E2 and P in sera and in blood from cord.Results: a) with increase of residual level of T-OCPs in the women, concentrations of FSH, E2, P in sera and FSH, LH, E2 in cord bood increased, respectively. In the puerperant sera, FSH increased from 0.83 mIU/ml in the control group to 2.27 mIU/ml in the high residual group, P from 264.04 ng/ml to 327.22 ng/ml, E2 increased from 41.80 ng/ml in the control to 54.99 ng/ml in the mid-residual group, respectively. In the cord blood, FSH increased from 1.41 mIU/ml in the control to 2.91 mIU/ml in the high residual group, LH from 0.60 mIU/ml to 2.07 mIU/ml, E2 increased from 12.53 ng/ml in the control to 24.03 ng/ml in the mid-residual group, respectively. Concentrations of LH in the venous sera and P in the cord blood, however, decreased from 1.47 mIU/ml to 0.63 mIU/ml, and from 804.47 ng/ml in the control group to 604.70 ng/ml in the high residual group, respectively. Significant differences were found among them (P < 0.05). b) Correlation analysis indicated that negative correlations were found between concentrations of T-OCPs in the high-residual group and E2, P in sera (r = -0.5158, P = 0.0238; r = -0.6491, P = 0.0026) . Positive correlation existed between FSH in sera and FSH, LH, E2 in cord blood, between LH in sera and P in cord, between P in sera and LH, E2 in cord. But there were negative correlations between FSH in sera and LH in cord blood , between LH in sera and FSH, LH in cord blood, between E2 in sera and FSH in cord blood, between P in sera and P in cord blood (P<0.05) .Conclusions: Residues of OCPs in body may disrupt concentrations of reproductive hormones in blood.Part V Effects of residues of OCPs in pregnant women on expressional abundances of α-ER,β-EP and GnRH mRNAin placenta and umbilical tissuesObjective: To study effects on expressional abundances of α-ER,β-EP and GnRH mRNA in placenta and umbilical tissues by DDT and BHC and their metabolites in puerperants.Methods: The studied individuals were the same in the Part Four. 71 tissue samples came from placenta and umbilical cord, respectively. Expressional abundances of α-ER,β-EP and GnRH mRNA in placenta and cord were determined by RT-PCR . Correlations by Spearman were analyzed between residue levels of T-OCPs in blood and the genes' expressional abundances in tissues, between concentrations of the hormones in blood and expressional abundances of the genes in tissues, and between expressional abundances of the genes in placenta and in cord.Results: a) Residues of OCPs in pregnant women could up-regulate expression of α-ER and β-EP mRNA. In placenta, expression of α-ER mRNA increased from 1.62 in the control to 2.42 in the high residual group, and β-EP from 0.36 to 0.68. In umbilical cord, α-ER improved from 1.49 to 2.14, and β-EP from 0.24 to 0.46. There were significant differences among the control, low-, mid-, high-residual groups (P < 0.05 ) . GnRH mRNA expressions in placenta were also up-regulated, but no dose-dependent effect was found. Expressional abundance of GnRH mRNA in cord was not determined in the present study. b) Correlation analysis indicated that there were negative correlation between residual levels of OCPs in blood in the low-group and expression of α-ER mRNA in placenta and cord (r= -0.4874, P = 0.0116; r = -0.4815, P = 0.0127) , respectively. And positive correlation was found between residual levels of OCPs and expression of GnRH mRNA (r = 0.4870, P = 0.0116) . It also suggested that α-ER and β-EP expressions were positive correlation in placenta and cord, while GnRH expression in placenta was negatively related to α-ER,β-EP in cord. There were positive correlations between α-ER in placenta and E2 in cord blood, between β-EP in placenta and FSH in puerperant blood, and FSH, E2 in cord blood, between α-ER in cord tissues and E2 in cord blood, between β-EP in cord tissues and FSH, P in puerperant blood, and E2 in cord blood. But negative correlation existed between P in cord blood and α-ER, β-EP in placenta and cord tissues, respectively (P<0.05) .Conclusions: Residues of OCPs in pregnant women may disrupt expression of some environmental response genes in placenta and umbilical cord.Part VI Effects of residues of OCPs in pregnant womenon expression of the 3 proteins in placentaObjective: To explore effects of residues of OCPs in puerperants on expressional abundances of α-ER, β-EP and GnRH protein in placenta.Methods: The studied individuals were the same in the Part Four. 71 placenta samples were collected to determine expressional abundances of α-ER, β-EP and GnRH protein by SABC immunohistochemistry. Correlations by Spearman were analysed between T-OCPs' residual concentrations in blood and the proteins' expressional levels in placenta, between the proteins and the genes mRNA expression in tissues, between expressional abundances of the proteins in placenta and concentrations of the hormones in blood.Results: a) Residues of OCPs in the women could induce expression of α-ER, β-EP and GnRH protein in placenta. Expressional abundances of α-ER increased from 128.80 in the control group to 149.70 in the high residual group, β-EP from 102.98 to 121.17, GnRH from 127.75 to 153.95, respectively. But there was only significant difference on GnRH expression among the control, low-, mid- and high-residual group (P < 0.05) . b) Correlation analysis indicated that T-OCPs level in blood in the low-residual group was negatively related to α-ER protein expression in placenta (r = -0.5326, P = 0.0124), but was positively related to GnRH protein expression (r = 0.6210, P = 0.0103) . Positive correlations were found between α-ER, β-EP mRNA and α-ER, β-EP protein, between GnRH protein and GnRH mRNA in placenta, respectively. However, negative correlation existed between GnRH protein and α-ER mRNA, β-EP mRNA in placenta, respectively, between GnRH mRNA and α-ER, β-EP protein in placenta. There were positive correlations between α-ER protein expression in placenta and concentration of P in sera, E2 in cord blood, between β-EP protein in placenta and FSH in sera, FSH and E2 in cord blood, respectively. But there were negative correlation between P in cord blood and α-ER, β-EP protein in placenta, respectively (P < 0.05 ) .Conclusions: Residues of OCPs in pregnant women may disrupt expression of α-ER, β-EP and GnRH protein in placenta.To sum up, concentrations of DDT, BHC and their metabolites were still high in the targeted population in the present study. Furthermore, o,p-DDT and γ-BHC were the main components in the puerperant blood. Therefore, we suppose that certain OCPs such as lindane and dicofol were still illegally used in this region. The joint action of p,p-DDE and β-BHC on the pregnant mice have shown estrogenicity in female, which could lead to feminination in their offsprings. Residues of OCPs in pregnant women could disrupt levels of reproductive hormones in blood, induce expression of certain enviromental respone genes such as α-ER,β-EP and GnRH in placenta and umbilical cord. Therefore, they may result in reproductive dysfunction and maldevelopment. However, its detailed mechanisms deserve further studies.This study possesses some new ideas as follow: a) we combined organically population study with animal experiment. Through epidemiological survey, the confounding factors effecting endocrine dirupting action of OCPs were ruled out, which improved reliability of the other expremental results. The present study indicated that residues of OCPs in pregnant women were probably concerned with increase of concentrations of FSH, E2, P in puerperant sera and FSH, LH, E2 in cord blood, and with decrease of levels of LH in sera and P in cord blood. At the same time, they could induce expression of α-ER, β-EP and GnRH gene (protein) in placenta and umbilical cord. b) For the first time, pregnant mice were co-incubated by p,p'-DDE and β-BHC to explore their reproductive and developmental toxicity. The results suggested that p,p'-DDE and β-BHC could decrease weights of the pregnant mice, increased of organ coefficient of uterus and intraluminal fluid, decreased nidation, increased concentrations of estradiol and progestone in sera in pregnant mice. Meanwhile, they could lessen the number of live foetus while increase dead foetus, absorbed foetus and featus deformities, increase AD and ratio of F/M in F1 generation, and induce expression of α-ER and GnRH mRNA while suppress β-EP in placenta.
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