| BackgroundChromium compounds are widely used in industry:Chromium is the most widely used adding element in steel to improve corrosion resistance. Chromium can be used for chrome plating, alloy production in metallurgy industry, as well as pigment, dyestuff, paint, rubber and ceramic manufacture. The industrial production and use of chromium compounds lead to the contamination of surface water and groundwater, so the people exposure to chromium through the water. Hexavalent chromium in the wastewater is highly toxic, which will seriously pollute the surroundings of rivers, soil and groundwater sources. Cr (VI) is a reproductive metal toxicant that can traverse the placental barrier and cause a wide range of fetal effects. Studies have shown that the embryo and fetus in mice and rats after exposure to hexavalent chromium can produce toxic effects, including pregnancy complications.Chromium has become one of the major environmental pollutants of the water, which will cause great harm to the environment if no effective measures to take. In recent years, with the rapid development of nanotechnology, nanomaterials have increasingly broad application prospects. Experimental studies have shown that NMH is used for the treatment of wastewater containing chromium, which gets good results, fast absorption and high adsorption. NMH recovered after roasting, after many times for the treatment of waste-water containing chromium.Nanomaterials have unique characteristics, which makes the toxicity of nanomaterials also demonstrate its uniqueness. In recent years, reproductive and developmental toxicity has increasingly become recognized as an important part of overall toxicology. Although the reproductive toxicology of nanomaterials has been reported, there has been no study on female reproductive toxicity and fetal toxicity resulting from NMH. Therefore, the present study investigated the effect of NMH on reproductive system and fetal development in ICR mice exposed by gavage for GD1-GD10and GD7-GD16respectively. It is significant whether NMH used to process wastewater containing chromium safely for pregnant women or not.ObjectiveThe model of phase of peri-implantation and organogenesis period were established to observe the weight change of pregnant mice, each organ weight, organ coefficient, fetal mice in length, tail length, and ossification points to evaluate the reproductive toxicity and embryotoxicity of NMH adsorbent. By detecting calcitonin (CT), parathyroid hormone (PTH) and growth hormone (GH) levels of pregnant mice serum, placental pathology structural changes of pregnant mice and vascular endothelial growth factor(VEGF),soluble vascular endothelial growth factor receptor(sFlt-1), placental growth factor (PLGF) of serum and placenta slurries of pregnant mice to further explore the mechanism of its toxicity. This study will provide a reliable basis for whether it is safe to pregnant women and the fetus for the use of NMH to process wastewater containing chromium.Methods1. Establishment of animal modelsDesign for the exposure:Pregnant mice were gavage administration to NMH (577.4336mg/Kg.bw), nanocomposite (583.7636mg/Kg.bw) and K2Cr2O7(6.33mg/Kg.bw) for one time everyday at GD1-GD10during phase of peri-implantation and GD7-GD16during organogenesis period. The day that the female mice mated successfully was identified as the first day of gestation(GD1). The gavage volume was0.1ml/10g body weight. Mice in the control group were were given a gavage of distilled water. Collection of the observation indexes:Mice were sacrificed on GD19. The body weights during the pregnancy were recorded. The embryo and uterus from each litter were harvested and weighted and the number of embryos on GD19in each litter was counted. The weights of uterus, ovary, liver, spleen, kidney and lung were taken down and then these tissues were frozen and preserved in-80℃refrigerator for further analysis.2. Measurement of the development indicators of fetal miceThe body length and tail length of the fetal mice were measured. After detecting of appearance of deformity, half of the fetal mice were added to Bouin’s fluid for the use of fetal mice visceral malformations examination, and the other half of the fetal mice for bone examination after alizarin red staining.3. Detection of CT, PTH and GH concentration in maternal serumConcentration levels of CT, PTH and GH in maternal serum were determined by enzyme-linked immunosorbent assay (ELISA). The detection of hormone levels was operated according to the instructions of the ELISA kits.4. Detection of maternal placental pathologyRespectively, we detected maternal placental pathology changes with the naked eye, light microscopy and electron microscopy.5. Detection of VEGF, PLGF and sFlt-1in maternal serum and placentaThe detection of VEGF, PLGF and sFlt-1in maternal serum and placenta slurries was the same as the methods used in part3.6. Statistical analysis methodA computerized statistical program (SPSS19.0) was used for statistical treatment. The datas were first analyzed by homogeneity test for variance, followed by one-way analysis of variance (ANOVA) if the variances were homogenous, and then the different toxic effects between groups were determined by Dunnett’s t test. If the variance were not equal, the data would be analyzed by a non-parametric analysis of variance (Kruskal-Wallis H test). Statistical significance was determined at level of α=0.05.Results 1. Maternal toxicity induced by NMH exposure during phase of peri-implantation and organogenesis periodWe observed the maternal body weight during pregnancy, and organic weights and indices of the two experimental models stated above. None of these indicators show any significant variety among four groups at the administered doses. Neither did weights and coefficients of reproductive organs show much distinction among four groups. These results suggest that the dose we used may be not enough to exert evident effects on the general condition of pregnant mice and their basic organs.2. Embryotoxicity induced by NMH exposure during phase of peri-implantation and organogenesis periodWe observed total number of fetal mice, live birth, and absorption fetal, and found little difference among groups. However, the indices of fetus development are significant:the average weight, height, and tail length of fetal mice, and nest of fetal mice weight were diminishing from the group of distilled water, NMH, nanocomposite, to potassium dichromate, indicating the greatest fetal developmental toxicity of potassium dichromate, the weaker one of nanocomposite, and the least one of NMH. The same trend presented in the ossification of fetal mice, which is indicated by the coccygeal vertebra ossification points, the second phalanx of forearms and hindlimbs ossification points, and the abnormality of ossification (rib hypoplasia, sternum adhesion).3. Effects of NMH exposure on concentration of hormonesAlthough PTH level show little distinction among four groups, CT and GH levels are lower in the groups of potassium dichromate and nanocomposite in the model of exposure during organogenesis period.4. Effects of NMH exposure on placenta structure and functionPlacenta weight and coefficient showed little variance among groups. We didn’t see any significant pathological changes in the maternal placenta via visual observation and light microscopy in any group. However, by electron microscopy, we discovered the phenomenon of placenta cell death in the group of NMH, nanocomposite and potassium dichromate in the two models. Here, we observed the decrease of VEGF level in groups of potassium dichromate and nanocomposite and the decrease of PLGF level in group of potassium dichromate in maternal serum, in the model of exposed during organogenetic period, and the level of sFlt-1did not show significant distinction among groups in either models. While in the placenta slurries, VEGF levels in all three groups other than control group are lowered significantly, and PLGF level is dropped only in group of potassium dichromate, in the model of exposed during organogenetic period, and the level of sFlt-1still present little variation.Conclusion1. Exposed to NMH, nanocomposite and potassium dichromate during phase of peri-implantation and organogenesis period, fetal toxicity, especially fetal development toxicity, is the most remarkable, indicated by the shrunken size and reduced ossification.Three substances in the exposure dose have small maternal toxicity effect, no significant teratogenic effects.2. Placenta occurs pathological damage in each exposure groups in two models, which may be an important mechanism of fetal mice developmental retardation and incomplete ossification.3. The period of organogenesis exposed to NMH, nanocomposite and potassium dichromate lead to dysfunction of the placenta, which showed VEGF and PLGF level significantly reduced.4. CT and GH levels are lower in the groups of potassium dichromate and nanocomposite in the model of exposure during organogenesis period, which may cause fetal mice growth retardation and reduced ossification.NMH has less impact on serum hormone levels. |
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