| Backgroud:Organotin compounds (OTs) has been widely used as painting in ship surface coating for preventing biofouling, pesticides, preservatives (wood, fiber and leather), industrial microbicides, plastic stabilizer, etc. Due to the wide application and long time existence in the environment, OTs has caused serious pollution on the global environment (especially water). Tributyltin (TBT) is one of the most important one of OTs, and it is considered as one of the most toxic chemicals in the environment intro-duced by man-made factors by far. TBT is a kind of certain Endocrine disruptor (ED), the existed studies have shown that TBT to the mammals can cause immunotoxicity, neurotoxicity and reproductive toxicity, etc. However, the dose of these study used is high, and many studies have shown that the damage of ED is not linear dose response relationship, some time the harm of low doses may be higher than the high doses. Our research group have studied and found that perinatal low doses of TBTCl can disturb the level of estrogen in mice testis, result in the decrease of sperm counts, the sperm motility reduced, the pathological changes of testicular tissue occurred in epididymis and the aromatase levels decreased in testis. While we have not known the mechanism of above changes up to now, so we take the progeny male mice as the main research object on the basis of previous research in this study, add the Diethylstilbestrol (DES) group as the positive control, study the influence of TBTCl on the pregnancy out-comes of the pregnant mice, the sex and body development of the progeny male mice and the sex hormone levels in the serum, and do the preliminary exploration on the mechanisms of which at the molecular and gene level. We hope our study can provide the reference for the study on TBTCl disrupting the human endocrine balance and its mechanism.Objective:To discuss the toxic effects of perinatal exposure to low doses of TBTCl to the pregnancy outcomes, progeny male mice growth and sex hormone levels in serum, and do a preliminary exploration on its mechanism.Methods:The pregnant KM mice are randomly divided into4groups,6in each group, they are given corn oil as the negative control group,10,100μg/kg TBTC1and5μg/kg DES as the positive control group by gavage from days6of gestation to the end of lacation(Postnatal21day, PND21). We infect one time every day at the same time and the dose is1ml/kg. At the time of GDO, GD6, PND1and PND21the weight of the mothers are weighed, and then we begin to observe and record the condition of the abortion and death of the infected mother. After the mother cub, we begin to observe and record the number of birth and the sex ratio. We also need to record the number of deaths of each group of the offspring in lactation. We weigh the body weight of the offspring on PNDO,1,2,3,4,6,7,14and21, and measure the distance between the anus and genitals (AGD) of the male mice. Beginning to examine the hair growing on PND6, the incisors drop-in on PND7, the eye opening on PND12, the testicular des-cent on PND13and the preputial separation on PND28, and we must record their age respectively.On PND22and100, respectively,10male offspring mice (The positive control group is8.) were selected in each group randomly and killed after weighed and trunk blood was collected. The blood was let stand at room temperature for2hours and then centrifugaing for the serum and save it. The testicles, epididymis, brain, liver, spleen, kidney and thymus (the ventral prostate and seminal vesicle also were taken on PND100) were separated and weighed for account of viscera coefficient. The testis were immediately put into liquid nitrogen for preservation at-80℃. Using the method of enzyme-linked immunosorbent assay measure the concentration of estro-gen (E2), testosterone (T), free testosterone (F-T), follicle stimulating hormone (FSH) and luteinizing hormone (LH) in serum on PND22and PND100. Adopting the Real-time PCR method to detect the relative transcript level of Cyp11al, Cyp17a, Hsd3b2, Hsd17b3and Cyp19gene mRNA in offspring mice testicular tissue on PND22. And the detection of the methylation pattern of CpG islands in promter region of cAMP response element binding protein, proteasome subunit alpha2and estrogen receptor beta gene who can control aromatase expression by bisulfite sequencing polymerase chain reaction (BSP).Results:Comparing with the positive control group, the dosage group’s abortion (death) rate of the pregnant mice is significantly decreased, while with the negative control group has an increasing trend. On PND21, the weight of the pregnant mice is signify-cantly higher than the control group, the body weight gain of the pregnant mice is significantly higher than the control group during PND1-21and GD0-PND21, and during GD6-PND21the body weight gain of the pregnant mice is significantly incr-eased compared with the negative control group in100μg/kg TBTCl group. The number of TBTCl group’s offspring has a downtrend compared with the control group. In100μg/kg TBTCl and the positive control group the mortality of mouse pup are significantly increased compared with the negative control group, but there is no statistical significance between the positive control group and the100μg/kgTBTCl group.The time of testicular descent of TBTCl group is significantly ahead of time compared with positive control group, but no statistical differences compared with negative control group, and the testicular descent time of the positive control group is significantly delayed compared with the negative control group. On PND0and1, comparing with the negative control group the NAGD is significantly reduced, at the same time the weight is also significantly increased in100μg/kgTBTCl, while there is only a tendency to reduce compared with control group, but there is no statistical difference on PND3,6,14and21. In100μg/kg TBTCl group the body weight of the offspring is significantly increased compared with negative control group on PNDO,1,2,6,7and21, the body weight gain is significantly increased compared with the negative control group during PND4-6, PND0-7, PND14-21, PND0-21. On PND22the viscera coefficient of testicular is significantly lower than the control group in100μg/kg TBTCl group; In TBTCl group the Viscera coefficient of Liver is signifi-cantly reduced compared with control group, while the weight and viscera coefficient of Kidney are significantly higher than negative control group and the weight and viscera coefficient of Spleen are also higher than the control group. On PND100, the testicular weight of TBTC1group is significantly increased compared with negative control group, but is significantly reduced compared with positive control group; in100μg/kgTBTCl group the viscera coefficient of Seminal vesicle is significantly lower than negative control group, but the liver is significantly higher; The viscera coefficient of Thymus was significantly decreased compared with negative control group, while was significantly increased compared with positive control group; The viscera coefficient of thymus of positive control group is significantly lower than negative control group.The measurement results of hormone in male offspring mice serum show that in100μg/kg TBTC1group the concentration of E2and FSH are significantly lower than the control group on PND22, and on PND100the serum E2levels is also significantly decreased compared with negative control group, while compared with positive control group their is no statistically difference, and the serum E2level of positive control group is also significantly reduced compared with negative control group.On PND22, the key enzymes in offspring testis tissue of steroid hormones mRNA relative quantitative results show that the mRNA expression amount of Cyp17a gene is significantly higher than the control group, Hsd3b2gene is signifycantly in-creased compared with negative control group, but their is no significant differences compared with positive control group in10μg/kg TBTC1group; the mRNA express-ion amount of Hsdl7b3and Cypl9genes are significantly lower than negative control group in100ug/kg TBTC1group, and the infected group’s mRNA expression amount of Cyp19gene has a tendency to reduce, while their is no significant different-ce compared with positive control group; the mRNA expression amount of Hsdl7b3gene and Cyp19gene in positive control group have a decreased trend compared with negative control group, but no statistical difference; Cypllal gene’s mRNA express-ion in the infected group is no statistical difference compared with the control group.On PND22, through the cloning sequencing comparison we find methylation only exists in the promoter region of cAMP response element binding protein (CREB) gene. However, the promoter region of the Ubiquitin/proteasome subunit alpha2and estrogen receptor beta gene do not find.ConclusionPerinatal exposure to low doses of TBTC1can result in the abortion of pregnant mice increased, lead to obesity and eat their own children. What’s more, it also makes the male offspring feminized and affects the growth and organ development. Through the study of the mechanism we find in perinatal low-dose TBTC1treatment can influe-nee the methylation happened in the promoter region of CREB, lead to the mRNA ex-pression levels of key enzymes of steroid hormone synthesis change, eventually lead to the serum hormone level disordered. |
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