Effects Of Benzo(a)Pyrene On Gene Expressions Of Junctional Proteins In Primary Sertoli Cell Culture

Benzo[a]pyrene (BaP), a representative polycyclic aromatic hydrocarbon (PAH), is commonly generated through the combustion of fuels, car exhaust, and other organic materials. It is widely distributed in the environment so people can be exposed to it from contaminated air, smoking, and food.B(a)P has been reported to have neurotoxicity, carcinogenicity, immunotoxicity and reproductive toxicity. Carcinogenesis and mutagenesis has been mainly focused on but reproductive toxicity has been researched recently. Exposure of B(a)P can lead to testicular atrophy, reductions of sperm count and sperm motility, and decrease of seminiferous tubules weight and testosterone levels. However, mechanism of male reproductive toxicity by B(a)P exposure is not thoroughly understood.The proteins that form Cell Junction (Junctional Proteins) in Sertoli cell play a key role in testicular function. They form Blood-Testis Barrier which creates a unique microenvironment for spermatogenesis. In addition, they play an important role in regulating proliferation, maturation and differentiation of the testicular cells.Junctional Proteins also mediate the exchange of signals, materials and energy between testicular cells. Many exogenous chemicals have been reported to damage Junctional Proteins. However, whether B(a)P exposure can affect the expression of Junction proteins is still unknown.The aim of present study is to investigate the direct effects of B(a)P on mRNA expression of Junctional Proteins in Sertoli cell, and to analyze the possible regulation of mRNA expression of Junctional Proteins by B(a)P exposure.Methods:Primary Sertoli cells culture were established by isolating Sertoli cell from21-day-old male SD rats following two-steps enzyme digestion method. The cell survival rate is more than94%and purity is more than95%in established culture system. The doses of B(a)P used in this study were0,5,10and25μmol/L. Semi-quantitative PCR was used to determine mRNA expression of Junctional proteins. Enzyme-labelled immunosorbent assay was used to determine the activities of caspass-3, MDA, SOD, CAT and GSHpx. Caspass-3inhibitor and Antioxidant were used to analyze the regulation by B(a)P induced oxidative damage and caspase-3induction. Results1Effects of B(a)P on Sertoli cell and mRNA expression of Junctional proteinsMorphological observation showed a morphologies change after exposure to B(a)P for12hours at dose of25μmol/L. Sertoli cells became slender and expanding of intercellular space.Although there was a declining trend for cell vitality but no statistical differences was showed by comparing with control group after exposure of B(a)P for6hours. With the increase of dose and culture time, there was a statistical differences at25μmol/L of B(a)P for12hours culture comparing with control group(P≤0.05).After exposure to B(a)P for6hours, mRNA of connexin43, connexin26, and occludin had a decline trends with the increase of dose of B(a)P through PCR analysis. The significant differences were appeared at dose of25μmol/L by comparing with control group(P≤0.05). mRNA expressions of ZO-1and N-cadherin had no statistical differences compared with control group. After exposure to B(a)P for12hours, The significant differences of connexin43, connexin26, and occludin mRNA expressions decreased significantly at10μmol/L compared with control group(P≤0.05). ZO-1mRNA expression decrease significantly at25μmol/L compared with control group(P≤0.05). N-cadherin mRNA expression had no statistical difference compared with control group. These results suggested that mRNA of connexin43, connexin26, and occludin were sensitive to B(a)P effects than that of ZO-1and N-cadherin.Results2Caspase-3may regulate the change of mRNA expressions of Junctional proteins which induced by B(a)PCaspase-3is considered to be an enzyme mostly associated with apoptosis. Caspase-3played an important role in the initiation and development of apoptosis.After exposure to B(a)P for12hours, caspase-3activity increased with a dose-dependent manner. Caspase-3activity increased significantly at10μmol/L and25μmol/L compared with control group(P≤0.05). When added caspase-3inhibitor as well as B(a)P at the same time, the induction of caspase-3activity by B(a)P at10μmol/L and25μmol/L was inhibited. Caspase-3activity in cells that was caspase-3activity added There were no significantly differences of Caspase-3activity between the cells that was added caspase-3inhibitor as well as B(a)P and controls that was not added any B(a)P. At the same time,the declines of connexin43, connexin26, occludin, and ZO-1mRNA expressions by B(a)P were also inhibited with no statistical differences by comparing with that of control group. These results suggested that caspase-3may play a role in regulation of mRNA expressions of Junction proteins which induced by B(a)P.Results3Oxidative damage induced by B(a)P affects the mRNA expressions of Junctional proteinsAfter exposure to B(a)P for12hours, both of reactive oxygen species (ROS) level and dialdehyde (MDA) level increased with the B(a)P dose increasing. ROS and MDA levels increased significantly at10μmol/L and25μmol/L of B(a)P exposed groups compared with control group(P≤0.05). Antioxidant enzymes activities declined with the B(a)P dose increasing.:the activities of SOD and GSHpx decreased significantly at25μmol/L compared with control group(P≤0.05). CAT activity decreased significantly at10μmol/L and25μmol/L compared with control group(P^0.05).After adding antioxidant-lycopene as well as B(a)P at the same time, the increasing of ROD and MDA activities by B(a)P was inhibited, and the declines of SOD, GSHpx and CAT activities were also inhibited. There was no significantly difference between the B(a)P+lycopene groups and control group. Meanwhile, the levels of mRNA expression of connexin43, connexin26occludin and ZO-1in B(a)P+lycopene groups were similar to control group with no statistically significant differences. These results suggested antioxidant can protect mRNA expression of Junction proteins from B(a)P damage.Results4Co-exposure of PCB169reinforced effects of B(a)P on sertoli cells and mRNA expressions of Junctional proteinsAfter co-exposure of PCB169(5μmol/L) and B(a)P (5μmol/L,10μmol/Land25μmol/L), increased significantly toxic effects were found in parameters of cell viability, MDA level, mRNA expressions of connexin43and occludin at25μmol/L after exposure to B(a)P for12hours compared with single exposure group(P≤0.05). In the other indexes, there were tendency to increase the toxic effects by Co-exposure but no statistical significant differences were showed.ConclusionsBaP exposure decreased cell viability and mRNA expression levels of junction protein mNRA in primary Sertoli cells. The induction of caspase-3activity and oxidative damage by BaP may play important roles.