| Objective: To explore in vivo exposure levels of cigarettes by measuring exposure biomarkers.To explore the reproductive toxicity of cigarette smoking(CS) on male rats. To explore the mechanism of epigenetic regulation on male reproductive toxicity.Methods: SD rats(160 male rats and 80 female rats) were divided into 15 cigarette exposure groups(the exposure concentration of which were 10, 20 and 30 nonfilter cigarettes/day, the exposure duration were 2, 4, 6, 8 and 12 weeks) and a control group(without CS exposure). Male rats in CS exposure groups were copulated with female rats as the ratio of 1:1 for one week before execution, newborn rats were observed for 21 days after birth. Male rats were sacrificed respectively at different time-points. The concentration of plasma nicotine and cotinine were measured by GC-MS/MS; the histomorphology of testes were observed; TUNEL assay was performed to detect the apoptosis of testes; the testicular caspase-3 activity was measured by immunohistochemical assay; the mRNA and protein expression of Apaf-1, caspase-9, Bim, Bcl-w, Bak and Ube2 B were detected by Real-time PCR and western blot; the survival rate and mortality of newborn rats were calculated; the activity of HAT and HDAC were evaluated; testicular microRNA(miRNA) profiles after CS exposure were investigated using miRNA microarray. Results:(1) CS exposed rats displayed decreased locomotor activity, ataxic gait, irregular respiration, nasal noise, and salivation. Rats in CS exposure groups had lower body weight, the reduction of body weight was time and dose related(P<0.05);(2) The concentration of plasma nicotine had no statistical differences among different exposure regimes(P>0.05), there was no interactive effect between exposure duration and exposure concentration(P>0.05);(3) The concentration of plasma cotinine in CS group was higher than control group(340±41.97 ng/ml), the increasing of plasma cotinine in CS groups was time-related(P < 0.05), exposure concentration and duration had synergistic effect on the level of plasma cotinine(P<0.05);(4) The testes of rats in CS exposed groups exhibited disturbance in diverse stages of spermatogenesis and disruption of tunica propria and basement membrane, the disruption of testicular histomorphology was time and dose related;(5) The apoptotic positive cells were increased in TUNEL staining linked to CS exposure duration;(6) Cytoplasmic positive cells of caspase-3 were found in immunohistochemical staining; compared with control group, the mean density of caspase-3 in CS exposed rats had significantly increased since the second week(P < 0.05), the IOD caspase-3 had significantly increased since the sixth week(P<0.01);(7) The testicular mRNA and protein expressions of Apaf-1 in CS exposed rats were up-regulated, the up-regulate of Apaf-1 mRNA expression in CS exposed rats was time-related; the mRNA and protein expressions of Apaf-1 in CS exposed rats which had 20 and 30 nonfilter cigarettes per day for 12 weeks were significantly increased compared with control group(P<0.05);(8) The testicular mRNA and protein expressions of Capase-9 in CS exposed rats were up-regulated and time-related(P<0.05);the mRNA and protein expressions of Capase-9 in CS exposed rats which had 20 and 30 nonfilter cigarettes per day for 12 weeks were significantly increased compared with control group(P<0.05);(9) The testicular mRNA and protein expressions of Bim in CS exposed rats were up-regulated, the up-regulate of Bim mRNA expression in CS exposed rats was time-related; the mRNA expressions of Bim in CS exposed rats which had 10 nonfilter cigarettes per day for 12 weeks were significantly increased compared with control group(P<0.05);(10) The testicular mRNA expression of Bak in CS exposed rats was down-regulated at the beginning of CS exposure, and been up-regulated since the eighth week; the up-regulate of Bak protein expression in CS exposed rats was time-related(P<0.01); the protein expressions of Bak in CS exposed rats which had 30 nonfilter cigarettes per day for 12 weeks were significantly increased compared with control group(P<0.05);(11) The testicular mRNA and protein expressions of Bcl-w in CS exposed rats were down-regulated, the down-regulate of Bcl-w protein expression in CS exposed rats was time-related(P < 0.01), exposure concentration and duration had synergistic effect on protein expression of Bcl-w(P<0.01); the protein expressions of Bcl-w in CS exposed rats which had 30 nonfilter cigarettes per day for 6, 8 and 12 weeks were significantly decreased compared with control group(P<0.05);(12) The testicular mRNA expression of Ube2 B in CS exposed rats was up-regulated at the beginning of CS exposure, and been down-regulated since the eighth week; the down-regulate of Ube2 B protein expression in CS exposed rats was time-related(P<0.05); the protein expressions of Ube2 B in CS exposed rats which had 20 and 30 nonfilter cigarettes per day for 8 and 12 weeks were significantly decreased compared with control group(P<0.05);(13) The reduction of newborn rats was time and dose related, the survival rate of newborn rats was significantly different among exposure regimens(P<0.05);(14) The activity of testicular HAT in CS exposed rats was increased and dose-related(P<0.05); the activity of testicular HAT in CS exposed rats which had 30 nonfilter cigarettes per day for 8 and 12 weeks were significantly increased compared with control group(P<0.05);(15) The testicular HDAC activity in CS exposed rats had significantly increased since the sixth week(P<0.05);(16) A total of 5 miRNAs were found to be associated with CS exposure, GO and KEGG pathway analyses of the predicted miRNA targets further illustrate the likely roles for these differentially expressed miRNAs in apoptosis of testis. Conclusion: The concentration of plasma cotinine can effectively reflect in vivo exposure levels of cigarettes; CS exposure causes reproductive toxicity in male rats, the mechanism of which is activation of mitochondrial apoptosis signaling pathway causes irreversible apoptosis of testis; CS exposure causes testicular histone acetylation and distinct miRNA profiles which indicates the effect of epigenetic regulation on reproductive toxicity induced by cigarette smoking. |
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