The Role Of MRP1 And PGC-1α On The Lead-induced Mitochondrial Toxicity In Testicular Sertoli Cells

Lead is a heavy metal that has been widely used in the manufacturing industry and mobilized in the environment Lead can be absorbed by the human body and accumulated in the bone, kidney, and reproductive system, causing adverse effects such as change of neurotransmitter levels, dysfunction of vitamin D metabolism, disorder of sex hormone secretion, dysfunction of lymphocyte, and decrease in offspring survival rates. It is known that lead contamination and lead poisoning have been recognized as a global public health problem. Epidemiological and experimental studies have shown that lead exposure could exert significant toxicity on the development of spermatozoa, leading to decreased sperm count and sperm motility as well as infertility. The hypothesis has previously been proposed that lead ions may pass through the blood-testes barrier and affect spermatogenesis, but the precise integrated mechanism of lead poisoning still need to be fully understood.It is known that the mitochondrion is a center of energy metabolism for eukaryotic cells, produces ATP for cell survival, and involves multiple molecular events such as oxidative stress and apoptosis. Our previous study indicated that lead can accumulate in the mouse Sertoli cell （TM4） and exert cellular cytotoxicity. However, lead-induced cytotoxicity could be attenuated through MRP1 （multidrug resistance associated protein 1）-glutathione interaction mediated lead efflux. It is reported that lead exposure has caused mitochondrial ultrastructure damage including cristae loss and swelling. Importantly, MRP1 also expresses in the mitochondrion in many tissues. We are therefore able to solve two issues that whether mitochondrial MRP1 possess the capacity of efflux in the TM4 cells, and what exact role mitochondrial MRP1 plays in lead-induced toxicity in TM4 cells. Multiple experiments confirmed that lead exposure increased reactive oxygen species （ROS） and inhibited the activity of antioxidant defense enzymes, leading to oxidative stress and cellular damage. Several antioxidants could attenuate the lead-induced cytotoxicity, and protect the mitochondrion against free radicals, suggesting that oxidative stress may be a mechanism of lead poisoning in the Sertoli cells. The PPARγ coactivator-la （PGC-la） is a key regulator involved in the process of mitochondrial genesis and energy metabolism. The coactivator PGC-la, which mainly resides in the nucleus or mitochondrion, regulates the energy balance via interacting with or co-activating several transcription factors. In addition, PGC-la also mediated cellular signalling such as oxidative stress and apoptosis. However, the role ofPGC-la in lead-induced oxidative stress and mitochondrial apoptosis is still to be determined.Here we investigated the potential role of MRP1 and PGC-1α in the lead-induced mitochondrial toxicity in the TM4 cells using several methods such as protein expression analysis, lentivirus-mediated transfection, ultramicroscopic morphology and so on. The data showed that MRP1 may be associated with lead accumulation of mitochondria, and PGC-la overexpression plays a limited role in protecting against lead-induced mitochondrial toxicity. This study represents an updated understanding of lead-induced male reproductive toxicity, and provides a new target for combating reproductive toxicity, and plays a promoted role in environmental health and public health.Objectives（1） To know the effect of lead exposure on mitochondrial ultrastructure and function at subcellular and molecular level in TM4 cells, and to discover the toxic effect of lead acetate on mitochondrion of TM4 cells.（2） To indicate the expression of MRP1 in the lead-induced mitochondrial toxicity in TM4 cells, and confirm whether MRP1 possesses transport activity and to investigate the relationship between mitochondrial MRP1 and lead accumulation in the mitochondria of TM4 cells.（3） To evaluate the role of PGC-la in lead mediated oxidative stress and mitochondrial apoptosis, and the relationship between PGC-la overexpression and lead-induced mitochondrial dysfunction （oxidative stress and apoptosis）;（5） To provide evidence for understanding lead-induced male reproductive toxicity.Methods（1） The functional mitochondria were obtained using mitochondrial extraction kit;（2） Mitochondrial protein extracts were prepared with lysis buffer, and followed by denaturation, electrophoresis, transmembrane to detect the level of MRP1.（3） The combination of mitochondrial extracts and calcein-AM were to evaluate the transport activity of mitochondrial MRP1. The atomic absorption spectrometry was performed to detect the lead content within mitochondrion after lead exposure.（4） The forward primer and reverse primer of PGC-la gene were designed and synthesized, and was amplified using PCR. The recombinant plasmids were generated by interacting gene fractions and lentiviral vectors. The positive clones were selected, sequenced, extracted. Cells were infected with recombinant plasmids, and selection with puromycin. The cell lines were identified using immunofluorescence, Real-time PCR, and Western Blotting.（5） Mitochondrial ultrastructure were observed using transmission electron microscope.（6） The level of cellular ATP and activity of succinate dehydrogenase were detected using commercially available kit. ROS levels were detected using flow cytometry and ELISA Reader.（7） Flow cytometry were also used to detected the lead-induced apoptosis in TM4 cells. The activity of caspase 9 and caspase 3 were detected using commercially available kit. Western blotting was performed to examine the expression of SIRT3 and release of cyto c.（8） All quantitative data represented a minimum of three independent experiments conducted in triplicates. The data were represented as the means ± SD, and analyzed by Statistical Product and Service Solutions software （SPSS 17.0）. Statistical analysis of two independent groups was compared using student t test. The data of multiple （>2 groups） independent groups were analyzed using One-way ANOVA, followed by Student-Newman-Keuls comparisons to assess the differences between groups. A P value less than 0.05 was considered statistically significant.ResultsPart Ⅰ （1） MRP1 can be expressed in the mitochondria of the TM4 cells; MRP1（-）TM4 cells reduced the expression of mitochondrial about 55.3% compared with control （P<0.01）. The intensity of Calcein’s fluorescence is increased by 173.1% in the mitochondria of MRP1（-）TM4 cells than in that of control, suggesting that MRP1 knockdown decreased the expression of mitochondrial MRP1 protein and reduced activity of transport. （2） The expression of mitochondrial MRP1 was increased followed by increasing concentration of lead acetate. The data showed a dose/time dependent change of mitochondrial MRP1 after lead exposure. The expression of mito-MRP1 induced by 80 μM lead acetate was 1-fold larger than control （P<0.01）. Lead exposure for 24 h and 48 h increased the expression of mitochondrial MRP1 by 59.4% and 79.9%, respectively, compared with control. （3） The results from atomic absorption spectrometry showed that the increasing mitochondrial lead content is followed by increasing lead concentration both in TM4 cells and MRPl-knockdown TM4 cells. In light of same dose of lead acetate, the lead content of MRP1（-）TM4 cells was higher than that of TM4 cells.Part Ⅱ The PGC-la gene fractions were successfully assembled through PCR amplification. Recombinant plasmids were obtained from enzyme digestion system in dependent on NotI/BamHI enzyme. The recombinant lentivirus was sequenced and compared with prototype sequences （NM₀08904）. The alignment data showed that recombinant sequences have a same result as prototype sequences. There was no significant base deletion or switching in the alignment data. These results showed that recombinant lentivirus vector is successfully generated. Fluorescence counting showed the titer of recombinant lentivirus vector is about 1.0×108 TU/ml. Expression results showed that the expression of PGC-1α mRNA increased by 2249.3%（P<0.01） compared with control, and the expression of PGC-1α protein increased by 89.4% （P<0.01） compared with control.Part Ⅲ （1） The data showed that lead acetate exposure change the expression of PGC-1α protein at various concentration. The PGC-1α protein from cell extracts exhibits an expression trend of first increase and then decrease, while mitochondrial PGC-la protein showed a decrease trend. （2） Ultramicroscopic morphology analysis showed that lead exposure causes mitochondrial ultrastructural damage such as broken cristae or less number of cristae, and mitochondrial swelling. Although lead acetate can worsen the mitochondria in PGC-1α（+）TM4 cells, the damage of mitochondrion in PGC-1α（+）TM4 cells is less than that in TM4 cells with exposure of same dose of lead acetate. （3） Lead exposure decrease ATP levels and activity of succinate dehydrogenase in a dose-dependent manner. Compared with control, the level of ATP increased about 7.3%～87.4% in exposure to 10 μM～80 μM dose of lead acetate. In light of the same concentration, the activity of succinate dehydrogenase is increased about 54.7%～161.8% in PGC-1α（+）TM4 cells than in TM4 cells. （4） In addition, lead exposure increased ROS levels in a dose-dependent manner. Flow cytometry analysis showed that the level of ROS is significant higher in TM4 cells than in PGC-1α（+）TM4 cells. These data suggest that lead exposure causes mitochondrial ultrastructure damage and oxidative stress, but higher expression of PGC-1α could attenuate these damage by lead. （5） Early apoptosis rates of TM4 cells and PGC-1α（+）TM4 cells increased with increasing lead acetate concentration. The exposure range from 20 μM to 80 μM lead acetate made TM4 cells increasing about 26.2%～64.8%. The apoptosis rate ofPGC-1α（+）TM4 cells reduced about 29.0%～37.1% than that of TM4 cells in the same concentration of lead acetate. （6） Lead exposure increased SIRT3 expression in TM4 cells to a maximum at 20 μM （P<0.01 vs control）, while lead-induced SIRT3 expression increased to a maximum （10 μM） in PGC-1α（+）TM4 cells. （7） The activity of caspase 9 and caspase 3 were increased by lead exposure, while caspase 9 activity of PGC-1α（+）TM4 cells reduced approximately 18.6%～32.3% and caspase 3 activity of PGC-1α（+）TM4 cells reduced approximately 8.5%-32.4%. （8） Western Blotting results showed that lead acetate induce an increase trend of the expression of cytoplasmic cyto c, and a decrease trend of the expression of mitochondrial cyto c at various concentrations. These data suggested that lead exposure promote the cyto c release from mitochondrion to cytoplasm. Compared with control, the expression of mito-cyto c in TM4 cells reduced about 50.7%, while the expression of mito-cyto c in PGC-1α（+）TM4 cells is reduced about 26.1%. Therefore, the relative release of mito-cyto c in PGC-1 a（+）TM4 cells was lower than that in TM4 cells. These data suggested that PGC-1α may be implicated in lead-induced apoptosis in TM4 cells, and these processes may be associated with cyto c release and change of caspase’s activity.Conclusions（1） Mitochondrial MRP1 possessed transport activity in TM4 cells. Lead acetate induced a dose/time effect on MRP1 expression in mitochondrion ofTM4 cells. MRP1 knockdown may increase the lead accumulation in the mitochondria.（2） The lentivirus-mediated PGC-la overexpression TM4 cell lines were successfully generated, which may be a helpful tool to evaluate the potential role of PGC-1α in Sertoli cells.（3） Lead exposures induced mitochondrial ultrastructural damage and dysfunction, and oxidative stress and apoptosis. PGC-1α overexpression plays a limited role in protection against mitochondrial toxicity in TM4 cells.