氟及氟铝联合对雄性大鼠生殖毒性的研究
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摘要
本研究通过建立动物模型的方法来探讨氟(F)及氟铝(Al)联合对雄性大鼠的生殖毒性及其可能机制。实验选择健康性成熟Wistar雄性大鼠56只,随机分为7组,每组8只,即：正常对照组(NS组),氟化钠(NaF) 1.0mg/kg、2.0 mg/kg、3.0 mg/kg组,NaF加铝离子(Al3+) 1.0 mg/kg＋0.1 mg/kg、2.0 mg/kg＋0.1 mg/kg、3.0 mg/kg＋0.1mg/kg组；称重,灌胃染毒,1次／天,连续90天。染毒结束次日,颈椎脱臼法处死大鼠,分别制备检测样本,进行下列实验：称重睾丸并计算睾丸脏器系数；精子质量检测系统测定精子运动参数；分光光度法检测睾丸组织匀浆中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)及一氧化氮合酶(NOS)的活性和丙二醛(MDA)、过氧化氢(H2O2)及一氧化氮(NO)的含量；原子吸收分光光度法检测睾丸组织中钙(Ca)、铁(Fe)、锌(Zn)、铜(Cu)和镁(Mg)的含量;蛋白免疫印迹法(Western blotting)检测睾丸细胞Fos蛋白的表达水平；流式细胞术检测睾丸细胞凋亡及其细胞周期的变化。研究结果如下：
1.与对照组比较,各实验组动物体重和睾丸脏器系数无显著性改变(P>0.05)。动物在给药过程中无死亡现象。
2.与对照组比较,各组精子的曲线速度(VCL)无显著性改变(P>0.05),高氟组、高氟加铝组精子平均路径速度(VAP)和高氟组鞭打频率(BCF)有显著性升高,低氟组前向性(STR)和侧摆幅度(ALH)有显著性降低,各组精子平均移动角度(MAD)和各氟染毒组直线性(LIN)均显著性降低,低、中氟组精子密度(P)显著性降低,高氟加铝组密度显著性升高(P<0.05)。同时,与高氟组比较,低氟组和中氟组精子P有显著性降低,高氟加铝组LIN有显著性升高(P<0.05)。各氟加铝组与对应氟染毒组比较,精子P均有显著性升高(P<0.01)。
3.与对照组比较,各组睾丸组织的SOD和CAT活性无显著性改变(P>0.05),低氟组MDA、中氟组H202、高氟加铝组NO含量均有显著性升高(P<0.05),低氟组NOS活性显著性降低(P<0.01)。同时,与相应氟染毒组比较,中氟加铝组H202含量有显著性降低,低、高氟加铝组NO含量有显著性升高,各氟加铝组NOS活性有显著性升高(P<0.05)。
4.与对照组比较,低氟组睾丸组织的Ca、中氟加铝组Fe、各氟染毒组和中、高氟加铝组Zn、高氟组Mg含量均有显著性降低(P<0.01)。同时,与低氟组比较,低氟加铝组Ca含量有显著性升高(P<0.01)。
5.与对照组比较,低、中氟组睾丸细胞Fos蛋白表达显著性增强(P<0.01)。与相应染氟组比较,各氟加铝组睾丸细胞Fos蛋白表达均显著性减弱(P<0.01)。
6.与对照组比较,各氟染毒组和低氟加铝组睾丸细胞G0/G1期均有显著性升高,各氟染毒组和高氟加铝组S期均有显著性降低,高氟组G2期有显著性降低,高氟加铝组G2期有显著性升高(p<0.05)。同时,与高氟组比较,高氟加铝组G0/G1期有显著性降低,G2期有显著性升高(P<0.01)。
7.与对照组比较,各氟染毒组睾丸细胞凋亡率均有所升高,其中中氟组和高氟组有显著性差异(P<0.05)。
本研究结果显示,过量氟暴露可引起雄性大鼠生殖的损伤,其机制可能与氟改变精子运动参数和睾丸组织金属元素含量、增强氧化应激效应及导致睾丸细胞Fos蛋白异常表达,进而引起睾丸生殖细胞周期紊乱、诱导生殖细胞凋亡有关。但是这种损伤改变并不随着氟剂量的升高而增强,在较低氟剂量水平表现更为明显,且氧化应激效应与细胞凋亡的改变并未呈现出一致的变化趋势。同时,在实验剂量范围内,铝对氟导致的生殖毒性有一定的拮抗作用,这可能是由于该剂量范围内的铝抑制了肠道对氟的吸收,增加了粪氟的排泄,从而拮抗了氟的生殖毒性。
This study investigated the toxic effects of fluoride (F) and combination of F and aluminum (Al) on reproduction of male rats and their possible mechanisms by establishing animal model. Fifty-six male Wistar rats with sexual maturation were divided into seven groups randomly, including control group with normal saline solution,1.0,2.0, and 3.0 mg sodium fluoride (NaF)/kg groups and each NaF-treated groups＋0.1 mg aluminium ion (Al3+)/kg groups with intragastric gavage for 90 days. The body and relative testes weights were calculated. The sperm motion parameters were measured by the WLJY-9000 color-detection system of sperm quality. The activities of superoxide dismutase (SOD), catalase (CAT) and nitricoxide synthase (NOS) and the contents of malondialdehyde (MDA), hydrogen peroxide (H2O2) and nitrogen monoxidum (NO) in the testis tissue were determined with spectrophotometric assay. The contents of calcium (Ca), ferri (Fe), zincum (Zn), cuprum (Cu) and magnesium (Mg) in the testis tissue were measured by atomic absorption spectrophotometry. The protein expression of Fos in the testis cell was detected by Western blotting. The testicular cell cycle and cell apoptosis were measured by flow cytometry. The results were showed as follows:
1. During the 90-day period of treatment and observation, there were no significant changes in body and relative testes weights between the control and any of the groups (p>0.05), and no death occured in any of the groups.
2. There were no significant differences in curvilinear velocity (VCL) of spermatozoa between the control and any of the groups (p>0.05). Significant increases were observed in velocity of average path (VAP) in the 3.0 mg NaF group and 3.0 mg NaF＋Al3＋group and beat cross-frequency (BCF) in the 3.0 mg aF group (p<0.05). Significant decreases in straightness (STR) and amplitude of lateral head displacement (ALH) of spermatozoa were observed in the 1.0 mg NaF group (p <0.05). There were significant decreases in mean angular deviation (MAD) in any of the groups and linearity (LIN) in all NaF-treated groups compared with the control group (p<0.05). In the 1.0 and 2.0 mg NaF groups, density (P) of spermatozoa was significantly lower compared with that in the control and 3.0 mg NaF groups (p <0.05). At the same time, significant increases in LIN in the 3.0 mg NaF＋Al3＋group andρin all NaF＋Al3+-treated groups were observed compared with the corresponding NaF-treated groups (p<0.05).
3. No significant changes in SOD and CAT activities in the testis tissue were observed in any of the groups (p>0.05). There were, however, significant increases in the level of MDA in the 1.0 mg NaF group, H2O2 in the 2.0 mg NaF group and NO in the 3.0 mg NaF＋Al3＋group and significant decrease in NOS activity in the 1.0 mg NaF group compared with the control group (p<0.05). At the same time, there were significant decreases in H2O2 in the 2.0 mg NaF＋Al3＋group and significant increases in NO in the 1.0 and 3.0 mg NaF＋Al3＋groups and NOS in all NaF＋Al3＋groups compared with the corresponding NaF-treated groups (p<0.05).
4. Compared with the control group, significant decreases were observed in the contents of Ca in the 1.0 mg NaF group, Fe in the 2.0 mg NaF group＋Al3＋group, Zn in the all NaF-treated groups and 2.0 and 3.0 mg NaF groups＋Al3＋groups and Mg in the 3.0 mg NaF group in the testis tissue (p<0.01). The content of Ca increased significantly in the 1.0 mg NaF＋Al3＋group compared with the 1.0 mg NaF group (p <0.01).
5. The protein expression of Fos in the testis tissue increased significantly in the 1.0 and 2.0 mg NaF groups compared with the control group (p<0.01), whereas decreased significantly in all NaF＋Al3＋groups compared with the corresponding NaF-treated groups (p<0.01).
6. Compared with the control group, significant increases in the percentage of G0/G1 stage cells in all the NaF-treated groups and in the 1.0 mg NaF＋Al3＋group and significant decreases in the percentage of S stage cells in all the NaF-treated groups and in the 3.0 mg NaF＋Al3＋group were observed (P<0.05). On the other hand, the percentage of G2 stage cells decreased significantly in the 3.0 mg NaF group, whereas inecreased significantly in the 3.0 mg NaF＋Al3＋group compared with the control group (p<0.05). At the same time, there were significant decrease in the percentage of G0/G1 stage cells and significant increase in the percentage of G2 stage cells in the 3.0 mg NaF＋Al3＋group compared with the 3.0 mg NaF group (p <0.01).
7. The testicular cell apoptosis ratios in all the NaF-treated groups were higher than that in the control group, but only the 2.0 and 3.0 mg NaF groups exhibited significant increases compared with the control group (p<0.05).
The results of this study indicated that excess F exposure has been found to produce adverse effects on the reproduction of male rats by changing the contents of metal elements in the testis tissue and the protein expression of Fos in the testis cell, inducing oxidative stress, changing testicular cell cycle and causing testicular cell apoptosis, especially at a comparatively lower level of exposure. However, the changes of testicular cell cycle and apoptosis do not appear to be directly connected with the increased level of oxidative stress, since some deleterious effects were more prominent at lower F intake. At the same time, Al has the antagonistic effect on the reproductive toxicity of F. The possible reason is that aluminum could inhibit the absorption and increase the excretion of F in intestinal tract.
1.与对照组比较,各实验组动物体重和睾丸脏器系数无显著性改变(P>0.05)。动物在给药过程中无死亡现象。
2.与对照组比较,各组精子的曲线速度(VCL)无显著性改变(P>0.05),高氟组、高氟加铝组精子平均路径速度(VAP)和高氟组鞭打频率(BCF)有显著性升高,低氟组前向性(STR)和侧摆幅度(ALH)有显著性降低,各组精子平均移动角度(MAD)和各氟染毒组直线性(LIN)均显著性降低,低、中氟组精子密度(P)显著性降低,高氟加铝组密度显著性升高(P<0.05)。同时,与高氟组比较,低氟组和中氟组精子P有显著性降低,高氟加铝组LIN有显著性升高(P<0.05)。各氟加铝组与对应氟染毒组比较,精子P均有显著性升高(P<0.01)。
3.与对照组比较,各组睾丸组织的SOD和CAT活性无显著性改变(P>0.05),低氟组MDA、中氟组H202、高氟加铝组NO含量均有显著性升高(P<0.05),低氟组NOS活性显著性降低(P<0.01)。同时,与相应氟染毒组比较,中氟加铝组H202含量有显著性降低,低、高氟加铝组NO含量有显著性升高,各氟加铝组NOS活性有显著性升高(P<0.05)。
4.与对照组比较,低氟组睾丸组织的Ca、中氟加铝组Fe、各氟染毒组和中、高氟加铝组Zn、高氟组Mg含量均有显著性降低(P<0.01)。同时,与低氟组比较,低氟加铝组Ca含量有显著性升高(P<0.01)。
5.与对照组比较,低、中氟组睾丸细胞Fos蛋白表达显著性增强(P<0.01)。与相应染氟组比较,各氟加铝组睾丸细胞Fos蛋白表达均显著性减弱(P<0.01)。
6.与对照组比较,各氟染毒组和低氟加铝组睾丸细胞G0/G1期均有显著性升高,各氟染毒组和高氟加铝组S期均有显著性降低,高氟组G2期有显著性降低,高氟加铝组G2期有显著性升高(p<0.05)。同时,与高氟组比较,高氟加铝组G0/G1期有显著性降低,G2期有显著性升高(P<0.01)。
7.与对照组比较,各氟染毒组睾丸细胞凋亡率均有所升高,其中中氟组和高氟组有显著性差异(P<0.05)。
本研究结果显示,过量氟暴露可引起雄性大鼠生殖的损伤,其机制可能与氟改变精子运动参数和睾丸组织金属元素含量、增强氧化应激效应及导致睾丸细胞Fos蛋白异常表达,进而引起睾丸生殖细胞周期紊乱、诱导生殖细胞凋亡有关。但是这种损伤改变并不随着氟剂量的升高而增强,在较低氟剂量水平表现更为明显,且氧化应激效应与细胞凋亡的改变并未呈现出一致的变化趋势。同时,在实验剂量范围内,铝对氟导致的生殖毒性有一定的拮抗作用,这可能是由于该剂量范围内的铝抑制了肠道对氟的吸收,增加了粪氟的排泄,从而拮抗了氟的生殖毒性。
This study investigated the toxic effects of fluoride (F) and combination of F and aluminum (Al) on reproduction of male rats and their possible mechanisms by establishing animal model. Fifty-six male Wistar rats with sexual maturation were divided into seven groups randomly, including control group with normal saline solution,1.0,2.0, and 3.0 mg sodium fluoride (NaF)/kg groups and each NaF-treated groups＋0.1 mg aluminium ion (Al3+)/kg groups with intragastric gavage for 90 days. The body and relative testes weights were calculated. The sperm motion parameters were measured by the WLJY-9000 color-detection system of sperm quality. The activities of superoxide dismutase (SOD), catalase (CAT) and nitricoxide synthase (NOS) and the contents of malondialdehyde (MDA), hydrogen peroxide (H2O2) and nitrogen monoxidum (NO) in the testis tissue were determined with spectrophotometric assay. The contents of calcium (Ca), ferri (Fe), zincum (Zn), cuprum (Cu) and magnesium (Mg) in the testis tissue were measured by atomic absorption spectrophotometry. The protein expression of Fos in the testis cell was detected by Western blotting. The testicular cell cycle and cell apoptosis were measured by flow cytometry. The results were showed as follows:
1. During the 90-day period of treatment and observation, there were no significant changes in body and relative testes weights between the control and any of the groups (p>0.05), and no death occured in any of the groups.
2. There were no significant differences in curvilinear velocity (VCL) of spermatozoa between the control and any of the groups (p>0.05). Significant increases were observed in velocity of average path (VAP) in the 3.0 mg NaF group and 3.0 mg NaF＋Al3＋group and beat cross-frequency (BCF) in the 3.0 mg aF group (p<0.05). Significant decreases in straightness (STR) and amplitude of lateral head displacement (ALH) of spermatozoa were observed in the 1.0 mg NaF group (p <0.05). There were significant decreases in mean angular deviation (MAD) in any of the groups and linearity (LIN) in all NaF-treated groups compared with the control group (p<0.05). In the 1.0 and 2.0 mg NaF groups, density (P) of spermatozoa was significantly lower compared with that in the control and 3.0 mg NaF groups (p <0.05). At the same time, significant increases in LIN in the 3.0 mg NaF＋Al3＋group andρin all NaF＋Al3+-treated groups were observed compared with the corresponding NaF-treated groups (p<0.05).
3. No significant changes in SOD and CAT activities in the testis tissue were observed in any of the groups (p>0.05). There were, however, significant increases in the level of MDA in the 1.0 mg NaF group, H2O2 in the 2.0 mg NaF group and NO in the 3.0 mg NaF＋Al3＋group and significant decrease in NOS activity in the 1.0 mg NaF group compared with the control group (p<0.05). At the same time, there were significant decreases in H2O2 in the 2.0 mg NaF＋Al3＋group and significant increases in NO in the 1.0 and 3.0 mg NaF＋Al3＋groups and NOS in all NaF＋Al3＋groups compared with the corresponding NaF-treated groups (p<0.05).
4. Compared with the control group, significant decreases were observed in the contents of Ca in the 1.0 mg NaF group, Fe in the 2.0 mg NaF group＋Al3＋group, Zn in the all NaF-treated groups and 2.0 and 3.0 mg NaF groups＋Al3＋groups and Mg in the 3.0 mg NaF group in the testis tissue (p<0.01). The content of Ca increased significantly in the 1.0 mg NaF＋Al3＋group compared with the 1.0 mg NaF group (p <0.01).
5. The protein expression of Fos in the testis tissue increased significantly in the 1.0 and 2.0 mg NaF groups compared with the control group (p<0.01), whereas decreased significantly in all NaF＋Al3＋groups compared with the corresponding NaF-treated groups (p<0.01).
6. Compared with the control group, significant increases in the percentage of G0/G1 stage cells in all the NaF-treated groups and in the 1.0 mg NaF＋Al3＋group and significant decreases in the percentage of S stage cells in all the NaF-treated groups and in the 3.0 mg NaF＋Al3＋group were observed (P<0.05). On the other hand, the percentage of G2 stage cells decreased significantly in the 3.0 mg NaF group, whereas inecreased significantly in the 3.0 mg NaF＋Al3＋group compared with the control group (p<0.05). At the same time, there were significant decrease in the percentage of G0/G1 stage cells and significant increase in the percentage of G2 stage cells in the 3.0 mg NaF＋Al3＋group compared with the 3.0 mg NaF group (p <0.01).
7. The testicular cell apoptosis ratios in all the NaF-treated groups were higher than that in the control group, but only the 2.0 and 3.0 mg NaF groups exhibited significant increases compared with the control group (p<0.05).
The results of this study indicated that excess F exposure has been found to produce adverse effects on the reproduction of male rats by changing the contents of metal elements in the testis tissue and the protein expression of Fos in the testis cell, inducing oxidative stress, changing testicular cell cycle and causing testicular cell apoptosis, especially at a comparatively lower level of exposure. However, the changes of testicular cell cycle and apoptosis do not appear to be directly connected with the increased level of oxidative stress, since some deleterious effects were more prominent at lower F intake. At the same time, Al has the antagonistic effect on the reproductive toxicity of F. The possible reason is that aluminum could inhibit the absorption and increase the excretion of F in intestinal tract.
引文
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