| BackgroundWith the rapid development of society and economy,nanotechnology has been rapidly developed,and nanomaterials have been widely used in all fields of life.Due to its unique antibacterial property,nanosilver(nAg)is one of the most widely used engineered nanomaterials.The widespread applications of nAg inevitably result in environmental release and human exposure,raising concerns about the health risks from exposure to nAg.The mammary gland is a susceptible organ to foreign materials.The usage of nanosilver in healthcare increases its risk of exposure to the mammary gland.The mammary gland provides nutrition to newborns and is an important route for newborns to expose nanomaterials.Exposure to foreign substances during mammary gland development may lead to mammary ductal hypoplasia and further affect the growth and development and immune function of offspring fed by breast milk.ObjectiveIn this study,BALB/c mice were used as animal models to investigate the effects of nAg exposure on mammary gland development during puberty.nAg particles with two sizes(20 and 50 nm)were selected to evaluate the contribution of particle size to the biological effects.Moreover,the potential effects of nAg on the lactating mammary gland were also scrutinized.We assessed the effects of nAg on the proliferation and apoptosis,and cell junctions of epithelial cells during lactating.Additionally,we clarified the distribution of nAg in the offspring and associated impacts on offspring growth.Method1.Toxicity and mechanism of nAg on mammary gland development in mice during pubertyIntraperitoneal injection was used to expose 4-week-old mice to nAg with different concentrations(5,20,100 μg/kg)and different particle sizes(20 nm,50 nm).Once every other day,each injection volume was 200 μl,and the inje ction was continuous for 3 weeks.After exposure,the content of silver in each tissue was measured by inductively coupled plasma mass spectrometry(ICP-M S).The level of alanine aminotransferase(ALT),aspartate aminotransferase(AS T),and malondialdehyde(MDA)in the serum was determined through immunoe nzyme-linked immunosorbent assay(ELISA).The mammary gland was spread onto a glass slide,fixed with 4% formaldehyde,stained with Carmine Alum,d ehydrated,and whole mounted with Permount.The level of estrogen receptor a nd ki67 on the mammary epithelial cells was measured through immunohistoch emistry techniques.2.Toxicity and mechanism of nAg on mammary gland development during lactationUsing tail vein injection,the female mice were exposed to different concentrations of 40 nm particle size,positively charged nAg(20,100,400 μg/kg).After exposure,The content of silver in blood of female and offspring mice was measured by inductively coupled plasma mass spectrometry(ICP-MS).The level of glutathione(GSH)and Progesterone in the serum was determined through immunoenzyme-linked immunosorbent assay(ELISA).The level of tight junction protein ZO-1 and proapoptotic protein Bax protein on the mammary gland epithelial cells was measured through immunohistochemistry techniques.Result1.nAg accumulated in the mammary gland after chronic exposureThe results of ICP-MS showed that nAg could enter the mammary gland by exposure.With the increase of the exposure concentration,the nAg entering the mammary gland increased.nAg with a diameter of 20 nm entering the mammary gland is 1.8 times that of nAg with a diameter of 50 nm.2.Pubertal exposure to nAg resulted in a decrease in the percentage of mammary ducts in the adult mice.The results of whole mounted showed that compared with the control group,the percentage of the distal ducts of the mice of 20 nm nAg(100 μg/kg)was significantlyreduced by 38.2%.3.nAg reduced the expression of estrogen receptor α and ki67 in pubertally exposed mammary epithelial cellsImmunohistochemical results showed that compared with the control group,the exposure of 20 nm particle size nAg(100 μg/kg)caused a 63.2% decrease in estrogen receptor α and a 56.2% decrease in ki67 expression in mammary epithelial cells.2.4.nAg induced adverse impacts on mammary glands at lactationImmunohistochemical results showed that compared with the control group,the expression of the proapoptotic protein Bax protein in mammary epithelial cells increased by 22.9%,and the expression of tight junction protein ZO-1 decreased by27.6%.The results of ICP-MS showed that when the female mice were exposed to nAg,the silver accumulation in the blood of the offspring mice,and the silver accumulation tended to increase with the increase of the concentration.5.nAg may affect the growth and development of the offspring miceThe analysis of the body weight of the offspring showed that the body weight of the 100 μg/kg nAg exposure group decreased by 17.9% compared with the control group,and the body weight of the 400 μg/kg nAg exposure group was 9.7% lower than the control group.This indicates that nAg exposure during lactation may significantly affect the growth and development of offspring mice.Conclusion1.nAg exposure can cause mammary growth retardation in puberty mice.2.Compared to the larger sized nAg,the smaller sized nAg was more likely to accumulate in the mammary gland,causing more significant adverse effects on mammary gland development during puberty.3.nAg induced cellular apoptosis of the mammary epithelium and disruption of cellular tight junctions between mammary epithelial cells.The disruption of cellular junctions resulted in the transduction of nAg to the offspring mice through milk feeding,which led to growth retardation in the offspring mice. |
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