| B(a)P is one of such pollutants threatening ecological environment and human health. It have been proved in lots of researches that B(a)P is a kind of human mutagen and carcinogen but little was known about the reproductive toxicity of B(a)P. This study was design to research reproductive developmental toxicity of B(a)P and its influences on parental generation and filial generation.In this study, testis tissues of male mice continuously exposed to B(a)P for 3 d and 7 d as the animal model. TBA colourimetry,KI colourimetry,SOD kit and OPA fluorometric method were applied to detect contents of MDA and H2O2, activities of SOD and GSH-Px; flow cytometry (FCM) was used to detect changes of cell cycle; CDNB colourimetry and RT-PCR were performed to examine GST activity and expression of GSTM1mRNA; fluorometric method,immunohistochemical method and Western Blot were applied to detect CYP1A1 activity and its protein expression; TUNEL was used to quantitatively detect apoptotic rate of germ cells; immunohistochemical method to detect the expression of apoptotic related proteins including AIF,Caspase-9,CytC,Bax and Bcl-2.1. Lipid peroxidation of testis cells of male mice induced by B(a)PIt was showed that 3 d after exposure, H2O2 content increased but MDA content decreased with the increase of B(a)P; 7d after exposure, contents of both MDA and H2O2 raised with increasing of B(a)P and there were significant differences in MDA content between 20,40mg/kg groups and control group (P<0.05) and H2O2 content between 10,20,40 mg/kg groups and control group (P<0.05).SOD kit and OPA fluorometric method were applied to examine activities of SOD and GSH-Px in testis tissues. It was showed the activities of SOD and GSH-Px decreased with increase of dosage and exposure period in both 3 d and 7 d of continuous exposure, there was significant difference between 40 mg/kg group and control group (P<0.05).2. Changes of cell cycle of testis cells of male mice induced by B(a)PAfter exposure to B(a)P for 3 days, cell percentage in G0/G1 phage unchanged but that in S phage decreased obviously, there were significant difference between different dose groups and control group (P<0.05); furthermore, cells in G2/M phage ascends in 10,20,40 mg/kg groups compared with control group (P<0.05). While after continuous exposure for 7 days, cells in G0/G1 phage descended and that in S phage increased.3. Changes of metabolic enzyme activities and the expression of GSTM1 mRNA of testis cells of male mice induced by B(a)PWith the increase of B(a)P and prolonged exposure period, the activity of GST and expression of GSTM1 mRNA depressed gradually and there was significant difference between 40 mg/kg group and control group (P<0.05) after 7 d of continuous exposure; the activity of CYP1A1 enzyme promoted and there was significant differences between 40 mg/kg group and control group (P<0.05) after both 3d and 7d of continuous exposure.4. Changes of expression of metabolic enzyme of testis cells of male mice induced by B(a)PImmunohistochemistry method was used. It could be observed under optical microscope that nucleus of positive expressed cells was dyed dark blue and cytoplasm or cellular membrane was dyed brown. With the adding of B(a)P, expression of CYP1A1 increased but there was no difference in 3 d groups and obvious differences between 10,20,40 mg/kg groups and control group after 7 d of continuous exposure (P<0.05).Furthermore, Western Blot was also applied for detecting expression of CYP1A1 induced by B(a)P. With the increase of B(a)P, the expression enhanced and got to the highest in 40 mg/kg group.Therefore, the same result could be concluded through the two different methods.5. Influence on cell apoptosis of testis cells of male mice induced by B(a)PAfter exposure to B(a)P for 3 d and 7 d, with the increase of dosage, cell apoptosis ascended to the highest in 10 mg/kg group and apoptotic rate descended with the prolonged exposure dose and period.6. Expression of apoptotic related genes of testis cells of male mice induced by B(a)PImmunohistochemistry method was used to detect expression of AIF,Caspase-9,CytC,Bax and Bcl-2. It could be observed under optical microscope that nucleus of positive expressed cells was dyed dark blue and cytoplasm or cellular membrane was dyed brown. After exposure to B(a)P (5,10,20 and 40 mg/kg) for 3 days, with the increased of B(a)P , the expression of AIF enhanced to the highest at first then weakened and there were significant differences between 5,10 mg/kg groups and control group (P<0.01); after 7 d of exposure, AIF expression decreased, there were obvious differences between 5,10,20 mg/kg groups and control group, respectively (P<0.05). After 3 d and 7 d exposure to B(a)P, Caspase-9 expression increased rapidly and then decreased with the adding of dosage and there were significant differences between exposure and control groups (P<0.05). After 3 d and 7 d exposure to B(a)P, CytC expression enhanced with the increase of B(a)P and there were significant differences between exposure and control groups (P<0.05). After 3 d and 7d exposure to B(a)P, Bax expression increased rapidly then decreased with the adding of dosage and there were significant differences between 5,10 mg/kg and control groups (P<0.05) in 3d of exposure while between 5,10,20 mg/kg groups and control group in 7d of continuous exposure (P<0.05).Generally, B(a)P could induce lipid peroxidation, decrease GST activity and expression of GSTM1 mRNA of testis tissues, increase CYP1A1 activity and protein expression leading to sever damage of testis cells. After continuous exposure to B(a)P for 3d, DNA synthesis of germ cells of male mice was depressed resulting in the occurrence of G2/M phage arrest; while in 7 d exposure groups, cells in G0/G1 phage decreased obviously and increased in S phage. B(a)P could also induce apoptosis of testis cells and changes expression of apoptotic related proteins including AIF,Caspase-9, Bax and CytC.
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