| BackgroundHexafluoroepoxypropane dimeric acid[(HFPO-DA),Gen X]is a new substitute for perfluorooctanoic acid(PFOA).Since 2010,it has been widely used as a fluoride processing aid in fields such as food packaging,fire protection,and interior decoration of buildings.HFPO-DA has been detected in various environmental media such as water sources and soil,suggesting that humans have been extensively exposed to HFPO-DA.But researching on its impact on human health is limited.Existing research suggested that HFPO-DA had endocrine disrupting effects,such as affecting thyroid function and leading to an abnormal lipid metabolism and glucose metabolism disorders.As one of the sex organs,the growth and development of the mammary gland is susceptible to various environmental endocrine disruptors,such as organochlorine pesticides,inorganic arsenic,and PFOA.However,there are no studies on the effect of HFPO-DA on the mammary gland.ObjectivesIn order to clarify the mammary developmental toxicity of HFPO-DA,in this study,we investigated the effect of HFPO-DA on the mammary gland development of pregnant rats and their offspring,and evaluated its interfering effect on the levels of mammary developmental hormones,and explored the related mechanisms.Methods1.Establishment a pregnant rat model exposed to HFPO-DA:healthy and sexual maturity SD rats were selected and caged according to the ratio of female to male 1:1.On gestational days 0.5(GD 0.5),SD rats were randomly divided into four dose-groups:control group(drinking deionized water),low-dose HFPO-DA exposure group(HFPO-DA gavage dose of 1mg/kg/d),medium dose HFPO-DA exposure group(HFPO-DA gavage dose of 10mg/kg/d),and high-dose HFPO-DA exposure group(HFPO-DA gavage dose of 100mg/kg/d),with 12 rats in each group.On GD19.5,six pregnant rats were randomly selected from each dose group for cesarean section,and the whole blood of pregnant rats and the fourth and fifth paired mammary glands and bilateral ovaries were collected.The fetal rats were removed to observe and record the number of live fetuses,the number of dead fetuses,the number of absorbed rats,and the presence of developmental malformations and finally retain the peritoneum of the fetal rats.The remaining 6 pregnant rats in each dose group were no longer subjected to HFPO-DA intragastric administration after GD19.5,and were delivered naturally.The litter number of pregnant rats and their weight,body length,tail length and anogenital distance(anal distance)were recorded on the postnatal day 1(PND 1).Their viability was recorded during PND 1-PND 20.The rats were sacrificed at PND 21 and cardiac blood collection was performed,and the fourth and fifth pairs of mammary glands and both sides of the female rats on PND21 were collected.2.Detection of mammary gland-associated hormone levels:Detecting the levels of Pg,E2,and PRL in the serum of pregnant rats and PND 21 offspring rats by ELISA to evaluate the endocrine disrupting effect of HFPO-DA on Pg,E2,and PRL.3.HE staining of ovaries:Taking ovaries of pregnant rats and PND21 rats for HE staining to evaluate the structural changes and development of ovaries.4.HE staining of mammary glands:Taking mammary glands of pregnant,fetal and PND 21 rats for HE staining to evaluate the structural changes and development of the mammary gland.5.Immunohistochemical staining of the mammary gland:A two-step method was used to assess the proliferation of the mammary gland.6.Quantitative analysis of mammary gland development indicators:Using the whole mount,whole tissue staining was performed on the mammary glands of fetal and PND21 rats to evaluate the development of the mammary gland.And quantitative analysis was conducted on the indicators of mammary gland development in fetal and PND21 rats.7.Statistical analysis:Using Image J,SPSS28.0 and Graphpad Prism8.0 software for measurement,data processing analysis and drawing.Measurement data were expressed as mean±SD(x±s),and one-way analysis of variance was used for comparison between multiple groups;the difference was considered statistically significant at P<0.05.Results1.Results of litter size and growth and development indicators on PND 1 rats:Litter size and PND 1 body length and tail length,weight and female and male anal distance were not significantly different in all groups.2.Results of mammary gland development-related hormones of PRL,E2 and Pg:(1)Pregnant rats:PRL concentration in serum of low-,medium-,and high-dose HFPO-DA exposure groups(4.16±0.81,4.58±1.13,5.66±1.32)were significantly higher than that in the control group(3.11±0.54).However,the difference was statistically significant only in the high-dose HFPO-DA exposure group(P<0.05).However,there was no significant difference in E2 and Pg concentrations in the low-,medium-and high-dose HFPO-DA exposure groups compared with the control group(P>0.05).(2)PND 21 rats:Compared with the control group,PRL,E2,and Pg concentrations in the low-,medium-,and high-dose HFPO-DA exposure groups were no significant difference(P>0.05).3.Results of the ovarian HE staining:(1)Pregnant rats:No significant differences in luteal structure and follicle morphology were observed in the low-,medium-,and high-dose HFPO-DA exposure groups compared with the control group.(2)PND 21 female rats:No significant differences were found in luteal structure and follicle development in the low-,medium-and high-dose HFPO-DA exposure groups compared with the control group.4.Results for mammary gland HE staining:(1)Pregnant rats:Large milk secretion was seen in the lumen and glands in the high-dose HFPO-DA exposed group compared with the control group.(2)Fetal rats:The number of mammary gland ducts increased significantly in the high-dose HFPO-DA exposed group compared with the control group.(3)PND 21 rats:The number of mammary gland ducts in the high-dose HFPO-DA exposure group was significantly increased,and the development of mammary gland secondary ducts was significant.5.Results of the immunohistochemical staining of the mammary gland:(1)fetal rats:compared with the control group,Ki67 staining in the low-,medium-and high-dose HFPO-DA exposure groups was significantly enhanced in a dose-dependent manner.(2)PND 21 rats:Ki67 staining of medium-and high-dose HFPO-DA exposed of females and males was significantly enhanced compared with control group,with no sex difference.6.Results of the whole staining of the mammary gland:(1)fetal rats:Quantitative analysis showed that duct coverage(788581.61±204442.21um~2),number buds of mammary gland ducts(8.71±1.11),and primary length(1042.37±126.96mm)in the 100mg/kg group increased significantly compared with the control group(466129.16±205902.91um~2,5.43±1.99,676.81±117.50mm,P<0.05).(2)PND 21 rats:The quantitative analysis showed that the mammary duct length of female ratsin the low-,medium-and high-dose HFPO-DA exposure groups(1807.74±54.11um,2014.82±23.61um,2162.24±37.17um)were significantly higher than that in the control group(1692.17±30.66um),and the difference was statistically significant(P<0.05).The duct length of male rats in the low-,medium-,and high-dose HFPO-DA exposure groups(1888.89±31.56um,1952.25±19.60um,2045.87±42.25um)were significantly higher than that in the control group(1820.02±32.36um),and the difference was statistically significant(P<0.05).Conclusions1.Exposure to HFPO-DA during pregnancy can increase the concentration of PRL in the serum of pregnant rats and induce the secretion of mammary gland milk.2.Exposure to HFPO-DA during pregnancy can induce the development of mammary gland by promoting the proliferation of mammary ductal cells in fetal and PND 21 rats,increasing the initial length,ductal coverage,the number of terminal buds and mammary gland duct length in PND 21 rats. |
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