| Objective:Melamine is considered a low toxicity of chemical raw materials, Animals, long-term intake of melamine will cause generation,urinary system damage, bladder,kidney stones, and further induce bladder cancer. Previous studies focused on the damage of animals' urinary system of melamine, the damage on the reproductice system were no systematic reported in domestic and international. In this study, we built a model of pregnant rats infected with continuous exposure to different doses of melamine in order to study:1. To investigate the effects of exposure to the persistent melamine on the placenta and fetus during gestation periods. 2. To investigate the effects of exposure to the melamine on the expression of gene Bax and Bcl-2 of gravid rats placenta cell apoptosis. Preliminary study the toxic effects and its mechanism of melamine to pregnant rats and its offspring.Methods:According to different dosage levels of melamine [0,400,800and 1200mg/(kg-d)], forty-eight gravid rats Wistar rats aged 3-month were randomly divided into control group, low-dosage group, mid-dosage group and high-dosage group, with 12 animals for each. All rats were orally fed with melamine from the sixth day of pregnancy in the morning 8-9 hours, and the capacity is 1mL/0.3kg weight, continuous for 14 days. The normal control group was given melamine-free vegetable oil. In the days of 0,6,11,16,20 of pregnancy, weighing the pregnant rats, according to the amount of body weight adjusted doses. Pregnant rats were anesthed by sodium pentobarbital at the 20th day.1. To take out the uterus and weigh lair weight, check uterus and count the number of nidation of amphicytula fetal death,merging fetal and vivi-fetal. The placentas and fetal body weight,body length and tail length were measured.2. We selected two fetus in every pregnant rat, taken out of the full fetal kidney. HE and electronic microscopy were used to observe the changes of fetal kidney morphology.3. HE and electronic microscopy were used to observe the changes of placenta morphology; Immunohistochemical method was used to examine the expression of gene Bax and Bcl-2 of gravid rats placenta cell apoptosis.4. All the datum were analyzed by statistics software SPSS 13.0. Group-samples T test was used to compare between four groups;χ2 analyses were used to compare frequencies between four groups.Results:1. There were no significantly differences in fetus'body weight,length and tail length between control group, low-dosage group, and mid-dosage group. The body weight,length and tail were the lowest in high-dosage group. There were significantly differences between the high-dosage group and the other three groups(P<0.05).2. There were no differences between the experimental groups and controls in placental weight.3. Lair weight was the lowest in high-dosage group. There were significantly differences between the high-dosage group and the other three groups(P<0.05). The number of the fetal death and merging fetal were increased in high-dosage group, vivi-fetal were decreased in high-dosage group. There were no differences between the experimental groups and controls in the nidation of amphicytula.4. There were no significantly changed in the structure of the fetus'kidney in control group and low-dosage group. Progeny of mid-dosage group and high-dosage group showed kidney crystalline material, there was inflammatory changed of the kidney in high-dose group.5. Changes of placenta morphology5.1 General observation:In the control group, the placenta was thick in center and thin in edge. The placenta was covered by transparent amniotic membrane, the surface was smooth; The placenta of low-dosage group and mid-dosage group were no significantly changed; the placenta of high-dosage group appeared purple,dull, some of the placenta were seriously congested, fetal amniotic surface was opacitas and luster dim.5.2 Electronic microscopy observation:The structure of baseband,areas spongiosa and labyrinth in control group were normal; trophoblast cells were no significant proliferation and villus was no swelling.There was no obviously changed in placenta structure of low-dosage group. The trophospongium cells were little degenerate of mid-dosage group, fibrin deposition around the villi was not evident. The placenta of high-dosage group showed focus necrosis in the baseband, and the trophoblastic giant cells and light staining cells were increased in the trophospongium. Trophoblast in the labyrinth and trophospongium showed degeneration; fibrin deposition around the villi was increased, tissues were seriously congested.6. The expression of Bax and Bcl-2 in placenta6.1 Bax and Bcl-2 all mainly expressed in syncytial trophoblast of placenta. Bax could also express in villi periplast cell and vascular endothelial cell, but Bcl-2 seldomly expressd in them. Bax mainly posited in endochylema and cell membrane; Bcl-2 mainly posited in endochylema, a few of Bcl-2 could also expressed in nucleus.6.2 There were significantly differences of Bax between the four groups(F= 17.42, P< 0.01). The expression of Bax gene in experimental groups was significantly higher than that in control groups. There were significantly differences of Bcl-2 between the four groups(F=23.62, P<0.01). There was a lower expression of Bcl-2 gene in experimental groups than that in control groups. And the ratio of Bax/Bcl-2 was higher in the experimental groups than that in control groups, there were significantly differences between them (F=66.65, P<0.01).Conclusion:1. High-dosage melamine can cause evidently embryo toxicity, showing fetus'body weight,body length and tail length were obviously degraded, the number of fetal death and merging fetal were increased, vivi-fetal were decreased. It indicated that high-dosage melanine may lead to retardation of fetus'normal growth and dystrophia.2. Exposure to persistent high-dosage melamine during pregnancy period may effect on fetus'kindey through the placenta, leading to geneogenous kidney disease.3. Exposure to persistent high-dosage melamine during pregnancy period may injure the trophocytes of the placenta, leading to the blood supply to the placenta and nutrition and oxygen exchange between rats and fetus were interfered, leading to retardation of fetus'normal growth and the other generation toxicity.4. Melamine could influence the expression of Bax and Bcl-2, leading to the increase of Bax,the decrease of Bcl-2 and increase the ratio of Bax/Bcl-2, damage the balance between cell proliferation and apoptosis. It may lead to placental dysplasia and produce harm to fetus. This change may be involved in the mechanism of rats generation toxicity and fetus growth retardation.
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