Low Dose Lead Exposure Disrupts Iron Homeostasis And Causes Abnormal Expression Of Transporter DMT1/FPN1 In Mouse

Objective:In recent year,low dose lead exposure at the onset of puberty exhibits its harmful effect on male reproductive system increased public health concerns.Dysregulation of testicular iron homeostasis is related to spermatogenesis,and causes diseases such as cryptorchidism and testicular cancer.However,the effect of long-term and low dose of Pb exposure on iron homeostasis is poorly understood.In this study,the deleterious effect of low dose Pb exposure on iron homeostasis is presented by the concentration of iron,and levels of m RNA and proteins of DMT1 and FPN1,furtherly provide scientific evidence for the mechanism of Pb toxicity.Methods:1.After a week of acclimatization,three weeks ICR mice were randomly assigned to different groups and drank distilled water containing 0,50 mg/L,and 200mg/L Pb acetate,calculated as Pb²⁺,for 90 days.2.Groups of five mice were housed in standard cages,and were free access to standard chow and distilled water ad libitum.Bodyweight of mice was recorded weekly.The mice were anesthetized with isoflurane at the end of Pb exposure（90 days）,and whole blood samples were collected from the heart and placed in ion-free tubes treated with 20%nitric acid for Pb analysis.The testes,small intestine,heart,liver,kidney and spleen were separated with plastic tweezers for elements analyses.3.（1）An analytical procedure for Pb determination via AA240FS-GTA120,were optimized by heating procedure and matrix modifier.（2）Determination of lead（Pb）,iron（Fe）,manganese（Mn）in whole blood and tissues（small intestine,liver,kidney,spleen,heart,testicles）.（3）The distribution of testicular iron in mice was observed by Perl’s staining.（4）The expression of testicular DMT1/FPN1 m RNA was detected by reverse transcriptase polymerase chain reaction（q RT-PCR）.（5）Western blotting（WB）was used to detect the expression of DMT1/FPN1 protein in testicular.Statistical analysis:SPSS17.0 software is used for statistical analysis.A one-way analysis of variance（ANOVA）was used to determine significant differences between groups.Statistical significance was set at P<0.05.Results:Optimization for lead determination by graphite furnace atomic absorption spectrometer（GFAAS）:the compromise pyrolysis and atomization temperatures were700℃and 1500℃,respectively.A higher lead absorbance value was obtained at the pyrolysis time of 30s.Then,addition of 5μL 1.0%ammonium dihydrogen phosphate（NH₄H₂PO₄）enhanced the absorbance values of these biological samples.Under the selected conditions,the recovery value was from 89.0%to 105.0%,which indicated a good accuracy.The achievable limit of detection（LOD）and limit of quantification（LOQ）values were 0.0949μg/L and 0.3164μg/L,respectively.The mass of body and testicular:the mass of body and testicular decreased after lead exposure.However,it had no statistically significant when compared with the control group.The elements levels of blood and tissues of mice:（1）Blood:the lead content was increased with Pb doses.The blood lead levels of 50 mg/L and 200 mg/L lead treated groups were（6.14±0.34 g/d L）and（11.92±2.92 g/d L）,and they were 8.71 and 16.93times as much as control group（P<0.05）.However,the iron levels of 50 mg/L and200 mg/L lead treated groups were（37.15±1.37 mg/d L）and（34.60±1.18 mg/d L）,and they were decreased by 11.0%and 17.1%compared with control group（P<0.05）.（2）Testicular:The lead content of 50 mg/L and 200 mg/L lead treated groups were（0.13±0.04μg/g）and（0.20±0.05μg/g）,and they were 6.67 and 10.17 times as much as control group（P<0.05）.The testicular iron level was increased with lead exposure levels.The iron content of 50 mg/L and 200 mg/L lead treated groups were（20.75±0.65μg/g）and（21.76±0.61μg/g）,and they were increased by 16%and 22%,compared with control group（P<0.05）.However,the manganese content was decreased with lead dose.The manganese content of 50 mg/L and 200 mg/L lead treated groups were decreased by 49.7%and 63.3%,respectively（P<0.05）.（3）Lead content of small intestine,liver,spleen,and heart in the 200 mg/L lead treated group were significantly higher than those of the control group（P<0.05）.The iron content of liver was increased significantly（P<0.05）,but it was decreased in kidney.The manganese content of spleen was decreased（P<0.05）.In the lead exposure group,the testicular was dispersed with granular blue staining,and the blue granules were mainly distributed in spermatidThe levels of iron transporter（DMT1/FPN）m RNA and protein expression in testis:level of DMT1 m RNA was up-regulated after Pb exposure;the amount of DMT1m RNA in 200 mg/L lead treated group was significantly higher than that in control group（P<0.05）.Level of FPN1 m RNA was up-regulated,but there was no statistically significant difference between groups（P>0.05）.（2）level of DMT1 protein was increased with lead doses,and DMT1 protein of lead treated groups was significantly higher than that in control group（P<0.05）.The level of FPN1 m RNA was down-regulated after Pb exposure.The level of FPN1 protein in 200 mg/L lead treated group was significantly lower than control group（P<0.05）Conclusions:Long-term and low dose lead exposure caused iron enrichment and disrupted transporter expression levels in testicular tissues.