低剂量铅暴露影响青春期小鼠睾丸铁稳态及转运体DMT1/FPN1表达的研究

Low dose lead exposure affects iron homeostasis and transporter expression of DMT1/FPN1 in puberty mice

目的 探究长期低剂量铅暴露对青春期雄性小鼠睾丸中铁稳态及其转运体DMT1/FPN1表达的影响。方法 4周龄SPF级ICR雄性小鼠按体重随机分为3组,每组15只。依据非职业人群血铅参考值及既往研究结果确定各组小鼠铅外暴露剂量分别为0(对照组)、50和200 mg/L,随饮水摄取。染毒90 d后,通过原子吸收光谱仪检测小鼠全血及睾丸中铅、铁含量;通过铁染色(Perl′s staining)法观察睾丸中铁的分布;采用qRT-PCR及Western blot检测睾丸中铁转运蛋白(DMT1/FPN1)mRNA及其蛋白表达水平;多组间数据比较采用单因素方差分析。结果 对照组、50和200 mg/L铅暴露组小鼠全血铅含量分别为(0.70±0.04)、(6.14±0.34)和(11.92±2.92)μg/dl,F血铅=31.937,P血铅=0.000,全血铁含量分别为(41.74±1.07)、(37.15±1.37)和(34.60±1.18)μg/dl,F血铁=15.867,P血铁=0.004,50和200 mg/L铅暴露组血铁含量均较对照组明显下降(P<0.05)。3组睾丸铅含量分别为(0.02±0.01)、(0.13±0.04)和(0.20±0.05)μg/g,F睾丸铅=5.011,P睾丸铅=0.026,睾丸铁含量分别为(17.83±1.18)、(20.75±0.65)和(21.76±0.61)μg/g,F睾丸铁=161.920,P睾丸铁=0.000,两个铅暴露组睾丸铁含量均较对照组明显上升(P<0.05)。Perl′s铁染色可见铅暴露睾丸中有明显蓝色颗粒分散分布,且主要分布在精细胞群中。睾丸中铁转运蛋白(DMT1/FPN1)mRNA及其蛋白表达水平结果显示,FDMT1mRNA=9.357,PDMT1mRNA=0.004;FFPN1mRNA=1.038,PFPN1mRNA=0.410;FDMT1蛋白=316.467,PDMT1蛋白=0.000;FFPN1蛋白=4.679,PFPN1蛋白=0.031。200 mg/L铅暴露组中睾丸二价金属离子转运体(divalent metal transporter 1,DMT1)mRNA表达水平明显高于对照组(P<0.05);膜铁转运蛋白(ferroportin 1,FPN1)mRNA表达水平有上升趋势,但各组间差异无统计学意义(P>0.05)。与对照组相比,50和200 mg/L铅暴露组DMT1蛋白表达水平均明显升高(P<0.05)。200 mg/L组睾丸组织中FPN1蛋白表达水平明显低于对照组(P<0.05)。进一步分析显示前期报道的精子密度、精子活率和精子畸形率与上述主要指标间统计学相关。结论 低剂量铅暴露能导致睾丸组织中铁富集及其转运体表达水平改变。

Objective To investigate the effects of long-term low dose lead exposure on iron homeostasis and transporter DMT1/FPN1 in testis of puberty male mice. Methods Four-week-old SPF ICR male mice were randomly divided into three groups according to body weight, with 15 mice in each group. According to the reference value of lead in blood of non-occupational population and previous result, the lead exposure dose of mice in each group was 0(control group), 50 and 200 mg/L with drinking water. After exposure for 90 days, the lead and iron contents of whole blood and testes were measured by atomic absorption spectrometer. Perl′s staining was used to observe iron distribution in testes. QRT-PCR and Western blot were used to detect the mRNA and protein expressions of DMT1/FPN1 in testes. Multi-group data were compared by one-way ANOVA. Results The lead contents of whole blood in control group, 50 and 200 mg/L lead exposure groups were(0.70±0.04),(6.14±0.34) and(11.92±2.92) μg/dl, respectively, Fblood lead=31.937, Pblood lead=0.000. The whole blood iron content was(41.74±1.07),(37.15±1.37) and(34.60±1.18) μg/dl, respectively, Fblood iron=15.867, Pblood iron=0.004.The lead content in testes of control group, 50 and 200 mg/L lead exposure group was(0.02±0.01),(0.13±0.04),(0.20±0.05) μg/g, respectively, Ftestes lead=5.011, Ptestes lead=0.026. Iron content in testes was(17.83±1.18),(20.75±0.65) and(21.76±0.61) μg/g, respectively, Ftestes iron=161.920, Ptestes iron=0.000. Compared with the control group, the blood iron content decreased and the testicular iron content increased in the two lead exposure groups, all the differences were statistically significant(P<0.05).Perl′s iron staining showed that there were obvious blue particles in the lead exposed testis, which were mainly distributed in spermatocytes. The mRNA and protein expressions of DMT1/FPN1 in testes show that FDMT1mRNA=9.357, PDMT1mRNA=0.004;FFPN1mRNA=1.038, PFPN1mRNA=0.410;FDMT1protein=316.467, PDMT1protein=0.000;FFPN1protein=4.679, PFPN1protein=0.031.The mRNA expression of DMT1 in 200 mg/L lead exposure group was significantly increased when compared with control group(P<0.05);the mRNA expression of FPN1 showed an upward trend, but there was no significant difference among all groups(P>0.05). Compared with the control group, the protein expression of DMT1 in 50 mg/L and 200 mg/L lead exposure groups was significantly increased(P<0.05).The protein expression of FPN1 in testes of 200 mg/L lead exposure group was significantly decreased when compared with control group(P<0.05).Further analysis showed that the previously reported sperm density, sperm motility and sperm deformity rate were significantly correlated with the above main indicators. Conclusion Low dose lead exposure could lead to iron overloaded in testes and alter the expression of its transporter.