| BackgroundLead was often present in people’s lives and work environment, it attached great importance in modern prenatal and postnatal care and male reproductive health problems since it was widely distributed, and could produce a male (male) sexual reproductive toxicity. Lead had effect on the male reproductive health, and could stay in testicular tissue through blood-testis barrier, thus caused direct toxicity and testicular tissue damage. Nrf2was a transcription factor in the CNC family member, was considered to be important intracellular oxidative stress regulon, was closely related with the antioxidant response element. Nrf2could regulate and control its downstream target gene regulation, and this includes some antioxidant enzymes Ⅱ phase detoxifying enzymes, drug efflux pumps, scavenger receptor and its transcriptional regulators by Keapl-Nrf2-ARE signaling pathway. MRPI was an ATP-dependent efflux pump, was regarded as representative of the glutathione-S-conjugated efflux pumps, and a variety of substances could be transported to the extracellular. Modern research had shown that each organism has its own defense system, certain heavy metals, such as mercury, cadmium and others could exert its own detoxification via Mrp1by Nrf2activation. Long et al study also showed that some of the toxic heavy metals, including lead, was able to induce MRPI gene transcriptional expression of the zebrafish.The testicular toxicity mechanism of Lead had still not fully understood, because of The side effects had been a lot of questions during of using of the drugs in Lead poisoning prevention process.It was great meaning to find out the mechanism of the toxic effects of lead and actively looking for an effective Excretion and effective treatment further. This study was carried out in vivo experiments and in vitro experiments to explore the the Nrf2and Mrpl impact of low lead on the testis of rat and the testicular Sertoli cells (TM4cells) of mouse, and look for the possible self-defensive Lead detoxification mechanisms; meanwhile it was adopted the quantitative analysis methods of meta-analysis of male reproductive health effects from lead in our country, to provide a theoretical basis for exploring new. more effective heavy metal detoxification pathways of lead.Part One:A Meta-analysis on the Effect of Lead on the Chinese Male Reproductive HealthObjective To polysynthetic analyze the effect of lead on male reproductive health. Materials and Methods Meta—analysis method was used to analyze the reported studies about the relationship between lead and male reproductive health. Results Common relative risk(RR) of male impotence and prospermia in the lead exposure group were2.55(95%CI=1.39～4.70) and2.23(95%CI=1.38～3.61) respectively, weighted mean difference (WMD) of the spermatic quantity, the spermatic deformity ratio and the spermatic activity ratio in the lead exposure group were-13.39×109/L (95%CI:-21.49×109/L～5.30×109/L),8.49%(95%CI:6.01%～10.97%)and-8.29%(95%CI:-12.94%～3.64%) respectively, which all showed significant differences between the two groups(P＜0.05). Conclusion The results indicated that lead relates to male reproductive health. It showed an increase in male impotence, prospermia and the spermatic deformity ratio, but showed a decrease in the spermatic quantity and the spermatic activity ratio.Part Two:The chronic effects of low lead level on the expressions of Nrf2and Mrpl of the testes in the ratsObjective The aim of this study was to explore the chronic effects of low concentrations of lead on the expressions of Nrf2and Mrp1in rats testes. Materials and Methods Female Sprague-Dawley rats were randomly assigned into3groups and administered lead acetate through drinking water freely from10days before gestation to weaning at the doses of0.0.8and1.5g/L, respectively; Then15male offsprings in each group were chosen and administrated lead acetate through drinking water freely from weaning to six months old at the doses of0.0.3and0.9g/L, respectively. RT-PCR. Western blotting, immunohistochemistry and immunofluorescence were used to detect the expressions or localization of Mrpl and Nrf2in testes. Atomic absorption spectrometry, spectrophotometry, enzyme linked immunosorbent assay were used to measure Pb2+concentration,GST activity and GSH content. Results As the lead dose rising. Pb2+concentration of rat testis were increased, GST activity and GSH content were decreased, both Nrf2and Mrpl expressions showed an ascending trend. The expressions of Nrf2and MRP1were consistent at the mRNA level and at the protein level. Significant increases were observed in the expressions of Mrpl among all groups (P＜0.05),and in the expressions of Nrf2between the two lead groups and the control group (P＜0.05). Immunofluorescence indicated the more lead was treated the more nucleus translocation of Nrf2was observed. Conclusion Mrpl might play crucial roles in cellular lead efflux by activating Nrf2.Part Three:The regulatory role of Mrpl expression by Nrf2in TM4cells of Low lead ExposureObjective The aim of this section was to further explore the detoxification defense mechanisms of the low lead exposed reproductive cells (TM4cells) by the treatments with tBHQ and ATRA. Materials and Methods The survival probability of TM4cell was detected after treating with tBHQ and ATRA by CCK-8assay. Created4model groups:the normal control group, the.20μM leadacetate treatment group, the20uM leadacetate+40μM tBHQ treatment group and20μM leadacetate of lead+2μM of ATRA group. Lead content within the cells in each group was determined by atomic absorption spectrometry, the expressions of Nrf2and Mrpl was detected by RT-qPCR, and the localization of Nrf2was examined by immunofluorescence. Results Compared with the normal control group, TM4cell viability of adding40μM tBHQ group was almost no significant difference (P＞0.05), TM4cell viability of adding2uM ATRA group was increased slightly, but no statistically significant (P＞0.05). The variation tendency of the expression of Mrpl was consistent to Nrf2in total. Compared with the first group, the expressions of Nrf2and Mrpl were increased in the other3groups, and had significant differences (P＜0.05). Compared with the second group, the expressions of Nrf2and Mrpl were increased in the adding tBHQ group, and Nrf2had a significant difference (P＜0.05) between them; while they were declined in the adding ATRA group, and Mrpl had a significant difference (P＜0.05). No significant nuclear translocation was observed, but Nrf2fluorescence intensity in the adding tBHQ group could be feeled increased than the others, lead content in cells of the normal control group is very low, lead content of the other groups were significantly increased (P＜0.05) when comparing with the control. Compared with the second group, lead content of the adding tBHQ group was reduced; while it was increased of the adding ATRA group, and had a significant change (P＜0.05).Conclusion The expression of Mrpl could be raised through the activation of Nrf2in TM4cells of Low Lead Exposure, thus it enhanced Mrpl efflux pump function and play cellular detoxification. |
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