The Reproductive And Developmental Toxicity Of Nano-iron Oxide And Its Related Mechanisms

Objective: The aim of this research is to evaluate the reproductive and developmental toxicity of nano-Fe₃O₄ particles in mice;the possible mechanism was investigated from the perspective of oxidative stress,P38 and Nrf2 gene regulation.The results can lay a theoretical basis for the safety assessment and application of nanomaterials in the future.Methods: 1,Transmission electron microscopy,laser particle size analyzer and X-ray diffractometer were used to analys the physical and chemical properties of nano-Fe₃O₄.2,60 male and 60 female mice?6 to 7 weeks old?were randomly divided into low?4.5 mg/kg BW?,middle?9 mg/kg BW?,high dose?45 mg/kg BW?nano-Fe₃O₄ treated groups and control group?normal saline treated only?,with 15 male or female mice in each group,respectively.The mice were exposed to nano-Fe₃O₄ by tail vein injection for 30 days.Sperm qualities of male mice and estrous cycle of female mice were measured.ELISA kits were used to detect the serum levels of follicle stimulating hormone?FSH?,luteinising hormone?LH?,prolactin?PRL?,estradiol?E2?,testosterone?T?and progesterone?PG?.Histopathological changes of the testes or the ovary were observed by the method of HE staining.Biochemical kits were used to determine the SOD activity and MDA content in the tissue.PCR was used to detect the m RNA expression levels of P38 and Nrf2 gene.The expression of Nrf2 protein in male testes was detected by the method of western blot.3,A total of 60 female mice?6 to 7 weeks old?were randomly divided into high dose?45 mg/kg BW?nano-Fe₃O₄ treated groups and control groups?normal saline only?.The mice were exposed at 15 days prior to mating to gestation day?GD?5,GD 5 to GD 15 and GD 15 to postnatal day?PND?21,respectively.The indicators as the number of live births,the number of stillbirths,the number of absorbing fetus,the weight of ovaries,the weight of placentas,the weight of living fetuses,the body length of living fetuses,the tail length of living fetuses,the appearance deformity,the bones deformity and visceral deformity were detected,respectively.Histopathological changes in the ovaries and placentas of pregnant mice were observed via HE staining.The rates of survival,the body development indicators?auricle separation,lower teeth eruption,eye opening and ear opening?and the reflective developmental indicators?plane turning,negative geotaxis,cliff avoidance and air turning?of offspring mice were detected.Results: 1,The nano-Fe₃O₄ particles has a diameter of about 20 nm,spherical structure,normal crystal and no impurities.2,The mice exposed to the particles grew normally,the sperm viability and motility of male mice in each dose group showed a decreasing trend,and the difference was statistically significant between the high dose group and the control group?P<0.05?.In the high dose group,the anestrus of female mice was significantly longer than that of control group?P<0.05?.With the increase of the dose,the testes and ovarian tissues of male and female mice showed varying degrees of pathological changes.The activity of SOD in the testes and ovarian tissues in the nano-Fe₃O₄ treated groups were significantly increased compared with that in the control group?P<0.05?.The content of MDA in the testes and ovarian tissues rose with the increasing doses of nano-Fe₃O₄,these differences were statistically significant when the male mice were treated at dose of 4.5 mg/kg BW or the female mice were treated at dose of 9 mg/kg BW?P<0.05?.There was no significant change of serum reproductive hormone levels in mice.The results of real-time quantitative PCR showed that the m RNA expression of Nrf2 in testis and ovary were decreased by nano-Fe₃O₄ treated,compared with that in control group,the m RNA expression of Nrf2 was significantly reduced in testis when the dosage reached 9 mg/kg BW?P<0.05?.The m RNA expression of P38 was slightly elevated,while the difference was not statistically significant compared to the control group?P>0.05?.The results of western blot showed that the content of Nrf2 protein in testis of nano-Fe₃O₄ treated groups were significantly higher than that of control group?P<0.05?.3,In the fertility and early embryonic developmental test,the rate of fetal resorptions in the nano-Fe₃O₄ treated group was increased significantly,whereas the weights and body lengths of the fetuses in the nano-Fe₃O₄ treated group were decreased significantly compared with those in the control group.In the embryo–fetal developmental test,placental weight decreased significantly in the nano-Fe₃O₄ treated group,but the other reproductive and developmental indicators showed no significant difference between the nano-Fe₃O₄ treated and control groups.Conclusions: The nano-Fe₃O₄ particles showed certain subacute effects on sperm activity of male mice and estrous cycle of female mice,it can induce the pathological changes and oxidative stress on the testis and ovary.There are no significant effects on the serum reproductive hormone of the mouse.The nano-Fe₃O₄ particles can affect the expression of Nrf2 gene in mice testes,but the mechanism of this effect needs to be further explored.The nano material has limited effects on pregnant mice and their offspring.Due to direct contact with human body,the bio-safety of this material also need to be further study.