Toxicity On Embryonic Developmental Induced By Carbon Nanotubes And Dibutyl Phthalate In Balb/c Mice

With the development of nanotechnology,carbon nanotubes(CNTs)have been widely used in various fields due to their characteristics.Due to its extremely small particle size,CNTs may enter the cells of animals by passive transportation.Studies have found that exposure to CNTs is toxic to the lungs,cardiovascular,liver,testis and other organs and tissues of animals.As one of the most commonly used plasticizers,dibutyl phthalate(DBP)is widely used in daily life,and it can enter the human body through a variety of ways.Studies have shown that DBP has a strong interference effect of environmental hormones,among which the reproductive toxicity is the most obvious.CNTs are one-dimensional carbon nanomaterials with a layered hollow structure.It is easy to form highly delocalized large π bonds between carbon atoms in the tubular structure.These π electrons can interact with other π electron-containing compounds through the π-π interaction.DBP is a compound based on aromatic benzene ring.In the environment,DBP is easily adsorbed by CNTs to form composite exposed pollutants.If CNTs adsorb DBP in the environment,carbon nanotubes will protect the DBP invisibly,and it will delay the degradation of DBP in the body.In this way,the endocrine disrupting effect of DBP will be prolonged,and the toxicity of DBP will increase.In this study,Balb/c mice were exposed to multi-wall carbon nanotubes(MWCNTs)and DBP individually and jointly.In order to explore whether MWCNTs as a carrier can delay the degradation of DBP and increase the endocrine disrupting effect of DBP,which will affect the development of mouse embryos.In this study,we previously studied the adsorption kinetics of short single-walled carbon nanotubes(SWCNTs)and short multi-walled carbon nanotubes(MWCNTs)on DBP.The results show that the fit of MWCNTs adsorption kinetics is higher than that of SWCNTS,so we decided to use MWCNTs for animal experiments.In animal experiments,we divided 40 female Balb/c mice and 40 male Balb/c mice randomly into 4 groups:(1)control group;(2)10 mg/kg MWCNTs group;(3)2.15 mg/kg DBP group;(4)MWCNTs+DBP group.Then,we infect mice by tail vein injection at a fixed time.After two weeks of exposure,the female and male mice were caged at a ratio of 1:1.The mice were exposed until the end of the 20th day of pregnancy in female mice.After the exposure,we put the female and male mice to death to carry out toxicological research:①We took out the embryos to check and count the embryonic development;② We took out the brain,spleen,liver and ovarian/testis tissues of female/male mice,and counted the body weight and organ-to-body ratio;③We analyzed the estrous cycle of female mice,sperm density and malformation rate of male mice;④We analyzed the oxidative stress related indicators of female mice ovary and male mice testis;⑤We analyzed histopathological changes in the ovaries of female mice and testes of male mice;⑥ We used mass spectrometry to analyze the differentially expressed proteins in the tissues of male and female mice;⑦We used RT-PCR to analyze Cyp11a1,Cyp17a1 and other related genes.The results showed that the embryonic mortality rate of mice exposed to MWCNTs and DBP was higher than that of mice exposed to MWCNTs and DBP alone.The results of oxidative stress and H&E staining showed that combined exposure caused ovarian tissue damage in female mice.And it caused the number of atretic follicles increases,and the number of other types of follicles decreases;Combined exposure caused pathological damage to the testis tissue of male mice,in which the number of spermatogenic cells in the testis tissue was reduced and the cell arrangement was disordered.Combined exposure can affect the estrous cycle of female mice.At the same time,it caused a decrease in sperm quality in male mice,but the difference was not significant compared with MWCNTs alone.We used mass spectrometry to analyze the protein expression in mouse serum and testis tissue,and the results showed that the expression of the protein encoded by the Hsd3b1 gene was significantly different in the combined group.Because the gene Hsd3b1 is involved in the synthesis of sex hormones,it is related to the reproduction and development of mice.In order to further explore the expression of genes,we used RT-PCR to analyse the level of gene expression.The results showed that compared with the control,the combined exposure caused a significant decrease in the expression levels of genes involved in the synthesis of sex hormones in the ovarian tissues of female mice.And compared with MWCNTs alone,combined exposure caused significant differences in the expression levels of genes involved in sex hormone synthesis in the ovarian tissue of female mice(P<0.01).Compared with the control,the combined exposure caused a significant increase in the expression levels of genes involved in sex hormone synthesis in the testis of male mice.However,compared with MWCNTs alone,the combined exposure caused no significant difference in the expression levels of genes involved in sex hormone synthesis in the testis tissue of male mice.Conclusion:Balb/c mice are exposed to the combined exposure of MWCNTs and DBP to increase embryonic mortality.We infer that MWCNTs may be used as carriers to adsorb DBP into mice.MWCNTs may delay the degradation of DBP and increase the endocrine disrupting effect of DBP and change the sex hormone synthesis in female mice,which can produce greater biological toxicity to embryos and increased embryonic mortality.