Effects Of Intravenously Injected Iron Oxide Nanoparticles As T₂-MRI Contrast Agents On Reproductive System In Male Mice

Objective:To determine whether exposure to IONPs would affect male reproductive system in ICR mice and to explore the possible mechanisms by which it occurs.Methods:IONPs were prepared by synthesizing iron oxide nanocrystals by pyrolysis and wrapped with poly（acrylic acid）（PAA）using chemical cross-linking method,and the IONPs were characterized and tested by magnetic resonance.To simulate clinical applications,IONPs were injected in a single pass through the tail vein of ICR mice.The systemic toxicity of IONPs was initially assessed by monitoring body weight and food and water intake in mice for 28 days.The effects of IONPs on the major organs were evaluated by measuring the iron ion content,histopathological changes,and liver and kidney function indices in mice at 1,3,7,14,and 28 days after IONPs injection.To evaluate the effects of IONPs injection on the reproductive system of mice,the sperm count,viability and survival rate,testicular/epididymal weight,organ indices,and histopathological damage were examined in mice 1,3,7,14,and 28 days after IONPs injection.To investigate the possible mechanisms of IONPs affecting the male reproductive system,the hormone levels of follicle-stimulating hormone,luteinizing hormone and testosterone,the protein expression of CDC2 and Cyclin B1,the protein expression and distribution of N-adherin and Occludin,the functional indicators of testicular support cells,and theα-GC activity of epididymis were measured,as well as the monitoring of cellular oxidative stress and apoptosis were also performed.Results:The synthesized IONPs were homogeneous in size and uniform in distribution,with good MRI contrast enhancement,demonstrating their potential as T₂-MRI contrast agents.The results showed that there were no significant changes in body weight and food and water intake in the mice treated with different doses of IONPs compared with each control group within 28 days after IONPs injection.IONPs mainly accumulated in the liver,spleen and lungs after entering the body via the tail vein of mice for 1 day,and almost rarely accumulated in the brain.No significant damage was observed in HE staining of major organs,and no significant abnormalities were observed in liver and kidney function indices,indicating no significant systemic risk to ICR mice after intravenous administration of IONPs.administration of IONPs resulted in a dramatic decrease in sperm count in mice starting 1 day after injection in a dose-dependent manner,and sperm quality was consistent with changes in sperm count on day 1 after IONPs injection.The sperm survival rate was significantly lower in the low dose group（28.56±1.14%）medium dose group（17.56±0.81%）and high dose group（8.75±0.32%）than in the control group（49.79±0.57%）,while the motility rate was extremely low in the high dose group（2.74±0.38%）compared to the normal control group（39.43±0.77%）.Monitoring of testicular structure and function showed no significant differences in testicular size,weight,shape,organ coefficients,hormone levels and expression levels of CDC2/Cyclin B1,an index of testicular function,between the groups.HE staining showed that apoptotic cells were observed in the testes of mice in the high-dose group on the first day after injection of IONPs,and the Johnsen score of the testes（9.17±0.31）was significantly lower than that of the control group（9.83±0.17）.Both Johnsen score and sperm parameters in the high-dose group gradually recovered within 2 weeks.IHC results showed that injection of IONPs reduced the local expression and density of N-cadherin and Occludin proteins,making the tight junctions between supporting cells discontinuous.The results of tests on epididymal structure and function showed no significant differences in epididymal weight,organ coefficient and epididymal function indexα-GCase activity between the groups.Iron ions could accumulate rapidly in the epididymis,reaching a maximum value of 44.15±5.20%ID/g1 hour after injection,while rapidly decreasing to a very low level（1.99±0.35%ID/g）within 24 h.HE staining results showed that on day 1 after injection of IONPs,almost no viable spermatozoa were observed in the epididymal lumen,and the sperm count was returning to the normal range on day 14 after injection.The sperm count was returning to normal range by day 14 after injection.The assessment of the degree of oxidative stress in the epididymis showed that the MDA level in the high-dose group（18.26±0.57 nmol/mgprot）was significantly higher than that in the control group（7.93±0.78 nmol/mgprot）,and the SOD activity（26.37±0.82 U/mgprot）was significantly lower than that in the control group（43.05±3.10 U/mgprot）.TUNEL staining showed apoptotic cells in the epididymal lumen of mice in the high-dose group,and dose-dependent positive staining for active caspase-3 could be observed by IHC staining.Conclusions:These results demonstrated that IONPs could cause reversible damage to the reproductive system of male mice without affecting the main organs,which may be due to increased oxidative stress and apoptotic activity in the epididymis.This result provides new guidance for the clinical application of IONPs as T₂-MRI contrast agents.