三氯异氰尿酸对SD大鼠睾丸结构和功能的影响
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摘要
随着工业的迅速发展,迄今为止已有大约10万种化学品进入人类的日常生活。人们在生产和生活中接触的许多种化学品都会影响人类的生殖功能,使人类的生育能力特别是精子的质量和数量发生显著改变。外来化学品所造成的生殖损伤日益受到人们的重视。
三氯异氰尿酸(Trichloroisocyanuric acid,TCCA)是一种新型、高效、广谱、低毒、性质稳定、效力持久、使用方便的消毒剂、漂白剂和防腐剂,广泛应用于饮食、医疗、卫生、农业、渔业、纺织、造纸等行业。国内外对TCCA的相关研究报道较少,仅有少量关于TCCA对单种生物的毒性研究,而其对人类生育能力的危害性尚未见到文献报道。
本研究以SPF级别SD大鼠为研究对象,通过TCCA较低剂量(0.71、2.29、7.59mg·kg-1·d-1)经口喂饲24月、较高剂量(96.5、193mg/kg)灌胃染毒10天和高剂量(275、550mg/kg)灌胃染毒10周三个实验,观察TCCA对雄性SD大鼠睾丸结构及功能的影响,明确TCCA对雄性SD大鼠生殖系统的损伤,从而确定TCCA的生殖毒性；采用免疫组织化学SABC法检测睾丸内凋亡相关蛋白Bcl-2/Bax、Caspase-3、FasL/Fas蛋白的表达；采用实时荧光定量PCR法检测睾丸内Bcl-2 mRNA和Fas mRNA|的表达水平；采用原子吸收光谱法检测睾丸内锌、.钙、铜的含量。通过这一系列的检测,较为深入的研究TCCA对SD大鼠睾丸毒性效应的作用机制,为进一步阐述TCCA的毒性作用机制奠定基础。
本研究的主要方法和结果：
1.TCCA对大鼠一般状态的影响：在0.71、2.29、7.59mg·kg-1·d-1剂量水平下连续染毒24个月,大鼠的一般状况与对照组相比均未见明显不同。在96.5、193mg/kg剂量水平下,染毒4天后大鼠出现活动减少,喜弓背行走,精神低靡的现象,但整个实验期间未出现大鼠的死亡。在275、550mg/kg剂量水平下,大鼠染毒后立即表现得比较烦躁,对外反应敏感,背部向上呈弯弓状,活动明显加强,好打斗；染毒4小时后大鼠活动明显减少,反应迟钝。染毒5周后,大鼠整日活动少或无活动,多以卧位为主,行动迟缓,进食量明显减少,毛发无光泽。染毒第9天开始出现大鼠的死亡,死亡大鼠肛周、唇部有血性分泌物。
以上实验结果提示：SD大鼠的一般状况(行为活动、饮食、生存、异常表现)随染毒剂量水平的高低明显不同。染毒剂量越高,毒性反应越明显,大鼠死亡出现的时间也越早。
2. TCCA对大鼠体重、生殖器官重量的影响
在0.71、2.29、7.59mg·kg-1·d-1剂量水平下连续染毒24个月,TCCA对各剂量组雄性SD大鼠的生长体重无明显影响,而对睾丸重量、睾丸脏器系数有明显影响。各剂量组的睾丸重量分别为3.009±1.206g、3.175±1.281g、3.232±1.800g,均低于对照组睾丸重量4.828±0.799g(P<0.001)。各个剂量组睾丸脏器系数分别为2.144±0.855、2.326±0.862、2.342±1.225,均低于对照组睾丸脏器系数3.603±0.462(P<0.001)。在96.5、193mg/kg剂量水平下,4和12周龄大鼠的体重增长均受到抑制；193mg/kg剂量水平下,4周龄大鼠睾丸重量(1.15±0.25g)和睾丸脏器系数(7.47±0.98)均低于对照组睾丸睾丸重量1.44±0.12g和睾丸脏器系数8.58±0.78(P<0.05)。在275、550mg/kg剂量水平下,TCCA可明显抑制大鼠体重,大鼠睾丸重量分别为3.40±0.44g、3.29±1.07g,均低于对照组睾丸重量3.58±0.62g(P<0.05)。
以上结果提示：随着TCCA染毒水平的增加,其对大鼠体重增长出现抑制作用,染毒水平越高,体重抑制现象越明显；长期接触较低浓度的TCCA或短期接触较高浓度的TCCA,均可降低睾丸重量,染毒水平越高,睾丸的重量减轻越明显。
3. TCCA对睾丸曲细精管直径的影响
在7.59mg·kg-1·d-1剂量水平下连续染毒12个月,SD雄性大鼠睾丸曲细精管直径为193.69±36.91μm,明显低于对照组大鼠睾丸曲细精管直径239.89±42.1μm(P<0.001)。在96.5、193mg/kg剂量水平下,4周龄大鼠睾丸曲细精管直径分别为167.30±12.55、144.23±11.35μm,明显低于对照组大鼠睾丸曲细精管直径183.82±20.36μm(P<0.01)；12周龄大鼠睾丸曲细精管直径分别为327.9±30.3、325.9±40.9μm,明显低于对照组大鼠睾丸曲细精管直径338.2±54.5μtm(P<0.01)。
以上结果提示：在一定剂量水平下,TCCA可使大鼠睾丸曲细精管变得细小,染毒水平越高,曲细精管越细小
4. TCCA对大鼠睾丸组织形态学的影响
在7.59mg·kg-1·d-1剂量组连续染毒12个月时,大鼠睾丸出现曲细精管萎缩、睾丸体积减小、精子弥漫性生成障碍等病变,对照组睾丸未检及以上病变。在0、0.71、2.29、7.59mg·kg-1·d-1剂量水平下染毒24个月时,各个剂量组各60例大鼠中,精子生成障碍分别为9、16、24、31例,曲细精管萎缩分别为12、16、23、32例,睾丸体积减小分别为8、15、20、31例,附睾腔内无精子分别为6、13、17、32；经统计分析,2.29、7.59mg·kg-1·d-1剂量组上述病理改变发生率均明显增高(P<0.001),说明在2.29、7.59mg-kg-1·d-1的剂量水平下,长期摄食TCCA会明显损伤大鼠睾丸的结构,影响精子形成,从而累及附睾内无精子,提示TCCA对雄性大鼠生殖系统表现出较为明显的毒性作用,主要的靶器官为睾丸。
在96.5、193mg/kg剂量水平下连续染毒10天,4周龄大鼠睾丸出现生精细胞退化(生精细胞核固缩)增加的现象,12周龄大鼠睾丸出现部分曲细精管生殖细胞层次紊乱、生精细胞脱落、支持细胞核空泡化、长形精子细胞退化等病理改变,且染毒水平越高,病理改变越明显。
在275、550mg/kg剂量水平下连续间隔染毒10周,大鼠睾丸出现曲细精管的局灶性、弥漫性萎缩以及个别曲细精管管腔不规则、支持细胞胞浆空泡化及间质细胞增生等病理改变,且染毒水平越高,病理改变越明显。
在193mg/kg剂量水平下连续染毒10天,TCCA可引起4、12周龄大鼠睾丸精原细胞核超微结构的改变,具体表现为：核膜的破损、核变形、染色质浓染等,但精原细胞未见明显的凋亡小体和坏死,也未见到其他细胞器在超微结构上的明显改变。
5. TCCA对大鼠睾丸细胞凋亡相关蛋白表达的影响
在275mg/kg剂量水平下,连续10周间隔染毒TCCA,采用免疫组织化学的SABC方法检测SD大鼠睾丸中凋亡相关蛋白Bcl-2、Bax、Fas、FasL、Caspase-3的表达情况,结果如下：Bcl-2阳性表达指数对照组为67.97%,275mg/kg剂量组为48.75%,Bcl-2表达降低(P<0.001); Bax阳性表达指数对照组为58.99%,275mg/kg剂量组为45.70%,Bax表达降低(P<0.001)；对照组睾丸曲细精管Bcl-2/Bax蛋白阳性表达的比值为1.15,275mg/kg剂量组Bcl-2/Bax比值为1.06,二者比值降低(P<0.05); Fas阳性表达指数对照组为82.31%,275mg/kg剂量组为78.46%,Fas表达指数无明显差异,但275mg/kg剂量组Fas表达强度明显增强；FasL阳性表达指数对照组为53.79%,275mg/kg剂量组为76.05%,FasL表达增加(P<0.001); Caspase-3阳性表达指数对照组为50.81%,275mg/kg剂量组为57.83%,Caspase-3表达降低(P<0.05)。
在275mg/kg剂量水平下,连续10周间隔染毒TCCA,采用荧光实时定量PCR的方法检测睾丸内Bcl-2和Fas mRNA表达水平,结果发现：对照组Bcl一2 mRNA相对表达数为0.062±0.015,275mg／kg剂量组相对表达数0.192±0.053,Bcl-2 mRNA表达明显升高(P<0.01)；对照组Fas mRNA相对表达数为0.072±0.005,275mg／kg剂量组相对表达数0.090±0.012,Fas mRNA表达明显升高(P<0.05)。
本次研究表明,TCCA可通过降低Bcl一2／Bax蛋白比值及增加Fas. FasL的表达,启动外源性、内源性凋亡途径,激活下游的效应Caspase-3,促使睾丸细胞凋亡的发生从而引起生精细胞脱落、数量减少,最终引发精子生成障碍、曲细精管萎缩。
6. TCCA对大鼠睾丸锌、钙、铜含量的影响
在275.550mg/kg剂量水平下,连续10周间隔染毒TCCA,采用原子吸收光谱法检测睾丸内锌、钙、铜的含量,结果发现：275.550mg/kg剂量组大鼠睾丸内钙含量分别为16.66±8.85 mg／kg.14.42±8.67 mg／kg,均低于对照组大鼠睾丸内钙的含量18.36±7.52mg/kg,但差异无统计学意义；275.550mg/kg剂量组大鼠睾丸内锌的含量分别为24.00±5.70 mg／kg.22.83±2.59mg／kg,均低于对照组睾丸锌含量24.41±7.88 mg／kg,其中550mg/kg剂量组大鼠翠丸锌含量降低有统计学意义(P<0.05)；275、550mg/kg剂量组大鼠睾丸内铜的含量分别为1.99±0.37 mg／kg.1.59±0.46mg/kg,均低于对照组睾丸锌含量2.03±0.87mg／kg,其中550mg/kg剂量组大鼠睾丸铜含量降低有统计学意义(P<0.05)。
7. TCCA对大鼠精子质量的影响
在96.5 mg/kg.193mg/kg剂量水平下12周龄成年大鼠连续10天染毒TCCA,通过检测附睾内精子数量、精子活动率及精子形态,评价TCCA对大鼠精子质量的影响。实验结果发现：对照组、96.5 mg/kg.193mg/kg组其精子数量分别为443.6±98.2、428.5±80.3和420.9±78.9×106/g,精子活动率均降低,分别为：82.9%、75.6%和64.84%,且染毒水平越高,大鼠附睾内精子数目和精子运动率降低越明显(P<0.05,P<0.01)。
采用伊红染色显微镜下观察大鼠睾丸精子形态,未见及精子头尾相连、卷尾、小头、无定型头、无头、无尾等几类畸形精子。
8. TCCA对SD血液系统的影响
在0.71、2.29、7.59mg·kg-1·d-1剂量水平下连续染毒24个月,分别于实验第6、12、18、24个月时,定期采集大鼠血液进行血液系统常规和血生化检测。结果发现虽然在某些检测时间点某数个检测项目与对照组相比增加或降低,差异有统计意义(P<0.05、P<0.01、P<0.001),但这种差异在各剂量组间的分布无明显规律性,未发现有明显的剂量-效应反应关系。表明在0.71、2.29、7.59mg·kg-1·d-1剂量水平下连续24个月染毒,TCCA对雄性SD大鼠血液系统无明显的毒性作用。
在96.5、193mg/kg剂量水平下连续10天染毒TCCA,4周龄幼年大鼠各个剂量组与12周龄大鼠96.5mg/kg剂量组大鼠血中AKP、ALT、AST、LDH的酶活性及血清离子Ca2+、Cl-、K+、Na+的浓度均无明显差异；12周龄大鼠193mg/kg剂量组血清LDH活性升高,血中Ca2+、Cl-降低(P<0.05),其他指标与对照组比较,差异均无显著性。
以上结果提示：TCCA对血液系统的影响与染毒水平有关,在在193mg/kg剂量水平下,TCCA能引起血液内LDH活性升高,Ca2+、Cl-浓度降低。
结论：
1、TCCA对大鼠的长、短期染毒均能导致睾丸组织学形态发生改变,并以睾丸重量减轻、睾丸曲细精管萎缩为主要改变,TCCA对雄性SD大鼠睾丸具有明显的生殖毒性。
2、TCCA对睾丸损伤程度与TCCA接触水平、接触时间呈量效关系,即睾丸的损伤程度随TCCA染毒水平的增加而加重,随接触时间的延长而加重。
3、TCCA可引起大鼠精子数量减少、精子运动率降低,但未发现其能导致精子畸形的发生。
4、TCCA促进睾丸生精细胞凋亡的发生是其毒性作用方式之一。睾丸细胞凋亡的增加与TCCA下调睾丸内Bcl-2/Bax蛋白表达、上调FasL和Caspase-3蛋白表达及Fas mRNA、Bcl-2mRNA表达水平有关。
5、TCCA可通过降低睾丸内锌、铜的含量,发挥其对睾丸的生殖毒性作用。
6、TCCA对大鼠行为活动、体重、血液系统的影响与染毒剂量水平有关。TCCA对雄性SD大鼠24月饲喂给药的最大无作用剂量为0.71mg·kg-1·d-1。若以100倍安全系数计,则其ADI为0.0071mg·kg-1·d-1。
With the speed development of industry, there are about 100 thousand chemicals in human daily life. It was proved that many kinds of chemicals could influence human reproductive function and make changes of human fertility particularly sperm count and quality. Peple had paid more and more attention to the damage of reproductive system by exotic chemicals.
Trichloroisocyanuric acid(TCCA) is a new, high-efficiency, wide-table, low-toxic, stable, effect-lasting, convenient disinfectant, which is already extensively applied in several industries(food profession,medical Industry,agriculture,fishery,textile and papermaking indu-stry). Although there have been some reports concerning on TCCA-induced toxicity of single species, few expriments on male reproductive toxicity and testicular injury were investigated.
This study here consists of three parts to determine the effect of TCCA on testicular strcture and function in SD rats, Exp.1 was to observe the long-term toxicity of TCCA in rats with various concentrations (0.71,2.29 and 7.59mg-kg-1·d-1) for 24 months.Exp.2 was to observe the short-term toxicity with the dosage of 96.5 and 193mg/kg through douche for 10 days and Exp,3 was to investigate the toxicity of TCCA and its molecular mechanisma with the dosage of 275 and 550 mg/kg through douche for 10 weeks (each other day)by the means of immunohistochemical method (SABC), real-time fluorescence quantitative PCR and atomic absorption spectrometry to detect the changes of Fas/FasL,Bcl-2/Bax-mediated apoptotic pathways, the contents of Zn,Ca and Cu in testis.
Main methods and results
1. Effect of TCCA on general conditions of Rats
A total of 270 rats,4 or 5 week-old, ingested TCCA in various concentrations (0.71,2.29,7.59mg·kg-1·d-1) for 24 months. And their general conditions, including behaviour, diet and drinking water had no significant difference compared with control group. When the rats were poisoned acutely through douche in the doses of 96.5,193mg/kg for 4 days, their physical activity decreased, back arched, and spirits sagged with no death in the experiment.When in the doses of 275,550mg/kg for 10 weeks,they immediately became sensitive to stimulus, too impatient and walked with the back arched.Then the rats had a dull reaction after 4 hours later and almost no activity after 5 weeks.Deaths happened with hemorrhagic secretions around oral and anal after 9 days.
These results suggested that the general conditions of rats exposed to TCCA varied with the dosage levels, meaning the higher the dosage of TCCA, the more obvious toxicity reactions, the early time of death.
2. Effect of TCCA on growth and reproductive organs weights of rats
In Exp.1, although the growth weights were not significantly lower in exposed rats than in control rats. the testicular weight loss occurred and organs index of testis decreased in exposed rats compared with the control rats. In Exp.2,the dosage of 193 mg/kg TCCA for 4 week-old rats could significantly decrease the weight and organs index of testis. And the dosage of 96.5 and 193mg/kg TCCA made the body weight loss both in 4 and 12 week-old rats. And in Exp.3, the body weight and testicular weight were also greatly decreased both in 275 and 550mg/kg groups.
All of the above demonstrated that the body weight of rats was significantly descend with dose increasing of TCCA,and exposed to TCCA long-time and low-dose or short-time and high-dose could cause weight loss of testis.
3. Effect of TCCA on Mean Seminiferous Tubules Dameter (MSTD) of testis
In this study, we observed the MSTD of testis with different concentration and operation time of TCCA.The results showed that the MSTD werel93.69,167.3,144.23,327.90 and 325.9μm correlated with dose of 7.59mg·kg-1·d-1,96.5 mg/kg in 4 week-old rat、193mg/kg in 4 week-old rat,96.5 and 193mg/kg in 12 week-old rat.All above MSDT were significantly declined compared with each controls and correlated with the dosage.These results suggested that TCCA could made seminiferous tubules thin and showed toxicity evidences of TCCA to testis.
4. Effect of TCCA on histomorphology of testis
The morphological changes of testis at 12 months characterized by atrophy of seminiferous tubules, arrest of spermatogenesis and smaller volume in 7.59mg-kg-1·d-1 goup rats,while there were no above-mentioned changes in control group. The incidence rates of above-mentioned changes at 24 month were significantly higher in 2.29 and 7.59 mg-kg-1·d-1 goup rats than that in control group. And the severity of histopathologic damage observed correlated positively with the increase of TCCA concentration and contact time. This histopathological examination suggested that TCCA could damag the structures of testis, cause disorder of spermatogenesis and make sperms disappear in epididymis. There was a obvious reproductive toxicity of TCCA and its primary target organ was testis in SD rat.
In the dose of 96.5 and 193mg/kg, the morphological changes of testis characterized by increased significantly degeneration of spermatogenic cells in 4 week-old rat,disorder and drop of spermatogenic cells, vacuoles in nucleus of Sertoli cells and degeneration of oblong spermatozoa in 12 week-old rat, the severity of histopathologic damage observed correlated positively with the increase of TCCA concentration
In the dose of 275 and 550mg/kg, the morphological changes of testis characterized by atrophy of seminiferous tubules, individual irregular tubular lumens, vacuoles in cytoplasm of Sertoli cells and noteworthy hyperplasia of testicular interstitial cells, and the severity of histopathologic damage observed correlated positively with the increase of TCCA concent-ration.
Additionally, in the dose of 193 mg/kg, the ultrastructure of spermatogonium was observed shown in detail as imperfect nuclear membrane, deformed nucleus, prominent clumped chromatin without clear apoptotic body and necrosis, while other cell organelles remained normal.
5. Effect of TCCA on expression of cell apoptosis associated ptoteins in testis of rats
In the study of toxic mechanisms induced by TCCA gavaged for 10 weeks (every other day), Immunohistochemical method was used to determine the expression of apoptosis associated proteins including Bcl-2,Bax,Fas,FasL,Caspase-3 and real-time fluorescenct quantitative PCR was uesed to carry on the quantitative analysis about gene expression of Bcl-2 and Fas mRNA.. The following results were obtained:
(1) Positive expression index of Bcl-2.Bax,Fas,FasL,Caspase-3 in control group and 275 mg/kg group were 67.97%/48.75%,58.99%/45.70%,82.31%/78.46%,53.79%/76.05% respectively, and there were significant differences between two groups except Fas.
(2) Bcl-2/Bax positive ratio in 275 mg/kg group were 1.06, which was significantly less than that in control group (1.15)
(3) There were higher positive intensity of Fas protein in 275 mg/kg group and significant difference in between two groups
(4) Compared with the control group, Bcl-2 and Fas genes were up-regulated in the 275 mg/kg group.
The results of this study suggest that apoptosis of spermatogenic cells can be induced by TCCA which may priming extrinsic and intrinsic pathways by up-regulating Fas/FasL and down-regulating Bcl-2/Bax proteins, in turn, activate the expression of downstream Caspase-3 proteins.
6. Effect of TCCA on the contents of Zn, Ca and Cu in testis of rats
We adopted atom absorption spectrophotometry technique to detect the contents of zinc (Zn), copper (Cu), and calcium (Ca) in testis and found that the contents of these three elements (Zn, Cu, Ca) in control,275and 550mg/kg groups were24.41/24.00/22.83 mg/kg, 2.03/1.99/1.59mg/kg,18.36/16.66/14.42 mg/kg respectively. The tendency of their contents was gradually decreasing with dose increasing, and in 550mg/kg group, there was significant difference compared with control group except the element of Ca.
7. Effect of TCCA on quality of rat's sperm.
We assessed sperm quality reduced by TCCA in a number of ways, for instance concentration, percentage of motile sperm and sperm shape.In the dose of 96.5 and 193 mg/ kg, the number of sperm was 428.5 and 420.9×106/g, and the percent of motile sperm was 75.6%and 64.84%, and there was no typical abnormal sperm observed. Compared to control group, both the number of sperm and the percent of motile sperm decreased with dose increasing,and there was significant difference between experimental and control groups. The final results confirmed that TCCA could significantly reduce quality of sperm.
8. Effect of TCCA on the Blood system of rats
After poisoned by TCCA for 6,12,18 and 24 months in the dosage of 0.71,2.29,7.59 mg·kg-1·d-1,the rat's blood were obtained to routine blood test and blood bio-chemical index examine. However there was no significant correlation between TCCA concentrations and every index of blood system. At the level of 195mg/kg for 10 days, the enzymatic activity of serum lactate dehydrogenase (LDH) activitie was increased and serum chloride, calcium level became lower. All of the above demonstrated that the effect of TCCA on blood system related to poisoned dosage. and the higher doses of TCCA administered, the more damage on blood system.
Conclusions
1. TCCA had obvious reproductive toxicity to testis and could cause pathological changes of testis with loss of testicular weight and atrophy of seminiferous tubules in the end.
2. The degree of testicular damage by TCCA proportionated to length of exposure and concentration. The higher doses of TCCA administered, the longer time of TCCA poisoned, and the more severity of damage on testis,
3. TCCA could significantly reduce the quality of sperm in rats,
4. As a reproductive toxic chemical, one of important toxic mechanism for TCCA was induced apoptosis of spermatogenic cells.
5. TCCA might make the reproductive toxicity to the testis. by means of reducing the contents of Zn and Cu,
6. The effect of TCCA on behaviour,body weight and blood system related to poisoned dosage.The maxial noneffective dose of TCCA was 0.71 mg-kg-1·d-1 in SD male rats and the acceptable daily intake(ADI) was 0.0071 mg-kg-1·d-1 with 100 times safety coefficient.
三氯异氰尿酸(Trichloroisocyanuric acid,TCCA)是一种新型、高效、广谱、低毒、性质稳定、效力持久、使用方便的消毒剂、漂白剂和防腐剂,广泛应用于饮食、医疗、卫生、农业、渔业、纺织、造纸等行业。国内外对TCCA的相关研究报道较少,仅有少量关于TCCA对单种生物的毒性研究,而其对人类生育能力的危害性尚未见到文献报道。
本研究以SPF级别SD大鼠为研究对象,通过TCCA较低剂量(0.71、2.29、7.59mg·kg-1·d-1)经口喂饲24月、较高剂量(96.5、193mg/kg)灌胃染毒10天和高剂量(275、550mg/kg)灌胃染毒10周三个实验,观察TCCA对雄性SD大鼠睾丸结构及功能的影响,明确TCCA对雄性SD大鼠生殖系统的损伤,从而确定TCCA的生殖毒性；采用免疫组织化学SABC法检测睾丸内凋亡相关蛋白Bcl-2/Bax、Caspase-3、FasL/Fas蛋白的表达；采用实时荧光定量PCR法检测睾丸内Bcl-2 mRNA和Fas mRNA|的表达水平；采用原子吸收光谱法检测睾丸内锌、.钙、铜的含量。通过这一系列的检测,较为深入的研究TCCA对SD大鼠睾丸毒性效应的作用机制,为进一步阐述TCCA的毒性作用机制奠定基础。
本研究的主要方法和结果：
1.TCCA对大鼠一般状态的影响：在0.71、2.29、7.59mg·kg-1·d-1剂量水平下连续染毒24个月,大鼠的一般状况与对照组相比均未见明显不同。在96.5、193mg/kg剂量水平下,染毒4天后大鼠出现活动减少,喜弓背行走,精神低靡的现象,但整个实验期间未出现大鼠的死亡。在275、550mg/kg剂量水平下,大鼠染毒后立即表现得比较烦躁,对外反应敏感,背部向上呈弯弓状,活动明显加强,好打斗；染毒4小时后大鼠活动明显减少,反应迟钝。染毒5周后,大鼠整日活动少或无活动,多以卧位为主,行动迟缓,进食量明显减少,毛发无光泽。染毒第9天开始出现大鼠的死亡,死亡大鼠肛周、唇部有血性分泌物。
以上实验结果提示：SD大鼠的一般状况(行为活动、饮食、生存、异常表现)随染毒剂量水平的高低明显不同。染毒剂量越高,毒性反应越明显,大鼠死亡出现的时间也越早。
2. TCCA对大鼠体重、生殖器官重量的影响
在0.71、2.29、7.59mg·kg-1·d-1剂量水平下连续染毒24个月,TCCA对各剂量组雄性SD大鼠的生长体重无明显影响,而对睾丸重量、睾丸脏器系数有明显影响。各剂量组的睾丸重量分别为3.009±1.206g、3.175±1.281g、3.232±1.800g,均低于对照组睾丸重量4.828±0.799g(P<0.001)。各个剂量组睾丸脏器系数分别为2.144±0.855、2.326±0.862、2.342±1.225,均低于对照组睾丸脏器系数3.603±0.462(P<0.001)。在96.5、193mg/kg剂量水平下,4和12周龄大鼠的体重增长均受到抑制；193mg/kg剂量水平下,4周龄大鼠睾丸重量(1.15±0.25g)和睾丸脏器系数(7.47±0.98)均低于对照组睾丸睾丸重量1.44±0.12g和睾丸脏器系数8.58±0.78(P<0.05)。在275、550mg/kg剂量水平下,TCCA可明显抑制大鼠体重,大鼠睾丸重量分别为3.40±0.44g、3.29±1.07g,均低于对照组睾丸重量3.58±0.62g(P<0.05)。
以上结果提示：随着TCCA染毒水平的增加,其对大鼠体重增长出现抑制作用,染毒水平越高,体重抑制现象越明显；长期接触较低浓度的TCCA或短期接触较高浓度的TCCA,均可降低睾丸重量,染毒水平越高,睾丸的重量减轻越明显。
3. TCCA对睾丸曲细精管直径的影响
在7.59mg·kg-1·d-1剂量水平下连续染毒12个月,SD雄性大鼠睾丸曲细精管直径为193.69±36.91μm,明显低于对照组大鼠睾丸曲细精管直径239.89±42.1μm(P<0.001)。在96.5、193mg/kg剂量水平下,4周龄大鼠睾丸曲细精管直径分别为167.30±12.55、144.23±11.35μm,明显低于对照组大鼠睾丸曲细精管直径183.82±20.36μm(P<0.01)；12周龄大鼠睾丸曲细精管直径分别为327.9±30.3、325.9±40.9μm,明显低于对照组大鼠睾丸曲细精管直径338.2±54.5μtm(P<0.01)。
以上结果提示：在一定剂量水平下,TCCA可使大鼠睾丸曲细精管变得细小,染毒水平越高,曲细精管越细小
4. TCCA对大鼠睾丸组织形态学的影响
在7.59mg·kg-1·d-1剂量组连续染毒12个月时,大鼠睾丸出现曲细精管萎缩、睾丸体积减小、精子弥漫性生成障碍等病变,对照组睾丸未检及以上病变。在0、0.71、2.29、7.59mg·kg-1·d-1剂量水平下染毒24个月时,各个剂量组各60例大鼠中,精子生成障碍分别为9、16、24、31例,曲细精管萎缩分别为12、16、23、32例,睾丸体积减小分别为8、15、20、31例,附睾腔内无精子分别为6、13、17、32；经统计分析,2.29、7.59mg·kg-1·d-1剂量组上述病理改变发生率均明显增高(P<0.001),说明在2.29、7.59mg-kg-1·d-1的剂量水平下,长期摄食TCCA会明显损伤大鼠睾丸的结构,影响精子形成,从而累及附睾内无精子,提示TCCA对雄性大鼠生殖系统表现出较为明显的毒性作用,主要的靶器官为睾丸。
在96.5、193mg/kg剂量水平下连续染毒10天,4周龄大鼠睾丸出现生精细胞退化(生精细胞核固缩)增加的现象,12周龄大鼠睾丸出现部分曲细精管生殖细胞层次紊乱、生精细胞脱落、支持细胞核空泡化、长形精子细胞退化等病理改变,且染毒水平越高,病理改变越明显。
在275、550mg/kg剂量水平下连续间隔染毒10周,大鼠睾丸出现曲细精管的局灶性、弥漫性萎缩以及个别曲细精管管腔不规则、支持细胞胞浆空泡化及间质细胞增生等病理改变,且染毒水平越高,病理改变越明显。
在193mg/kg剂量水平下连续染毒10天,TCCA可引起4、12周龄大鼠睾丸精原细胞核超微结构的改变,具体表现为：核膜的破损、核变形、染色质浓染等,但精原细胞未见明显的凋亡小体和坏死,也未见到其他细胞器在超微结构上的明显改变。
5. TCCA对大鼠睾丸细胞凋亡相关蛋白表达的影响
在275mg/kg剂量水平下,连续10周间隔染毒TCCA,采用免疫组织化学的SABC方法检测SD大鼠睾丸中凋亡相关蛋白Bcl-2、Bax、Fas、FasL、Caspase-3的表达情况,结果如下：Bcl-2阳性表达指数对照组为67.97%,275mg/kg剂量组为48.75%,Bcl-2表达降低(P<0.001); Bax阳性表达指数对照组为58.99%,275mg/kg剂量组为45.70%,Bax表达降低(P<0.001)；对照组睾丸曲细精管Bcl-2/Bax蛋白阳性表达的比值为1.15,275mg/kg剂量组Bcl-2/Bax比值为1.06,二者比值降低(P<0.05); Fas阳性表达指数对照组为82.31%,275mg/kg剂量组为78.46%,Fas表达指数无明显差异,但275mg/kg剂量组Fas表达强度明显增强；FasL阳性表达指数对照组为53.79%,275mg/kg剂量组为76.05%,FasL表达增加(P<0.001); Caspase-3阳性表达指数对照组为50.81%,275mg/kg剂量组为57.83%,Caspase-3表达降低(P<0.05)。
在275mg/kg剂量水平下,连续10周间隔染毒TCCA,采用荧光实时定量PCR的方法检测睾丸内Bcl-2和Fas mRNA表达水平,结果发现：对照组Bcl一2 mRNA相对表达数为0.062±0.015,275mg／kg剂量组相对表达数0.192±0.053,Bcl-2 mRNA表达明显升高(P<0.01)；对照组Fas mRNA相对表达数为0.072±0.005,275mg／kg剂量组相对表达数0.090±0.012,Fas mRNA表达明显升高(P<0.05)。
本次研究表明,TCCA可通过降低Bcl一2／Bax蛋白比值及增加Fas. FasL的表达,启动外源性、内源性凋亡途径,激活下游的效应Caspase-3,促使睾丸细胞凋亡的发生从而引起生精细胞脱落、数量减少,最终引发精子生成障碍、曲细精管萎缩。
6. TCCA对大鼠睾丸锌、钙、铜含量的影响
在275.550mg/kg剂量水平下,连续10周间隔染毒TCCA,采用原子吸收光谱法检测睾丸内锌、钙、铜的含量,结果发现：275.550mg/kg剂量组大鼠睾丸内钙含量分别为16.66±8.85 mg／kg.14.42±8.67 mg／kg,均低于对照组大鼠睾丸内钙的含量18.36±7.52mg/kg,但差异无统计学意义；275.550mg/kg剂量组大鼠睾丸内锌的含量分别为24.00±5.70 mg／kg.22.83±2.59mg／kg,均低于对照组睾丸锌含量24.41±7.88 mg／kg,其中550mg/kg剂量组大鼠翠丸锌含量降低有统计学意义(P<0.05)；275、550mg/kg剂量组大鼠睾丸内铜的含量分别为1.99±0.37 mg／kg.1.59±0.46mg/kg,均低于对照组睾丸锌含量2.03±0.87mg／kg,其中550mg/kg剂量组大鼠睾丸铜含量降低有统计学意义(P<0.05)。
7. TCCA对大鼠精子质量的影响
在96.5 mg/kg.193mg/kg剂量水平下12周龄成年大鼠连续10天染毒TCCA,通过检测附睾内精子数量、精子活动率及精子形态,评价TCCA对大鼠精子质量的影响。实验结果发现：对照组、96.5 mg/kg.193mg/kg组其精子数量分别为443.6±98.2、428.5±80.3和420.9±78.9×106/g,精子活动率均降低,分别为：82.9%、75.6%和64.84%,且染毒水平越高,大鼠附睾内精子数目和精子运动率降低越明显(P<0.05,P<0.01)。
采用伊红染色显微镜下观察大鼠睾丸精子形态,未见及精子头尾相连、卷尾、小头、无定型头、无头、无尾等几类畸形精子。
8. TCCA对SD血液系统的影响
在0.71、2.29、7.59mg·kg-1·d-1剂量水平下连续染毒24个月,分别于实验第6、12、18、24个月时,定期采集大鼠血液进行血液系统常规和血生化检测。结果发现虽然在某些检测时间点某数个检测项目与对照组相比增加或降低,差异有统计意义(P<0.05、P<0.01、P<0.001),但这种差异在各剂量组间的分布无明显规律性,未发现有明显的剂量-效应反应关系。表明在0.71、2.29、7.59mg·kg-1·d-1剂量水平下连续24个月染毒,TCCA对雄性SD大鼠血液系统无明显的毒性作用。
在96.5、193mg/kg剂量水平下连续10天染毒TCCA,4周龄幼年大鼠各个剂量组与12周龄大鼠96.5mg/kg剂量组大鼠血中AKP、ALT、AST、LDH的酶活性及血清离子Ca2+、Cl-、K+、Na+的浓度均无明显差异；12周龄大鼠193mg/kg剂量组血清LDH活性升高,血中Ca2+、Cl-降低(P<0.05),其他指标与对照组比较,差异均无显著性。
以上结果提示：TCCA对血液系统的影响与染毒水平有关,在在193mg/kg剂量水平下,TCCA能引起血液内LDH活性升高,Ca2+、Cl-浓度降低。
结论：
1、TCCA对大鼠的长、短期染毒均能导致睾丸组织学形态发生改变,并以睾丸重量减轻、睾丸曲细精管萎缩为主要改变,TCCA对雄性SD大鼠睾丸具有明显的生殖毒性。
2、TCCA对睾丸损伤程度与TCCA接触水平、接触时间呈量效关系,即睾丸的损伤程度随TCCA染毒水平的增加而加重,随接触时间的延长而加重。
3、TCCA可引起大鼠精子数量减少、精子运动率降低,但未发现其能导致精子畸形的发生。
4、TCCA促进睾丸生精细胞凋亡的发生是其毒性作用方式之一。睾丸细胞凋亡的增加与TCCA下调睾丸内Bcl-2/Bax蛋白表达、上调FasL和Caspase-3蛋白表达及Fas mRNA、Bcl-2mRNA表达水平有关。
5、TCCA可通过降低睾丸内锌、铜的含量,发挥其对睾丸的生殖毒性作用。
6、TCCA对大鼠行为活动、体重、血液系统的影响与染毒剂量水平有关。TCCA对雄性SD大鼠24月饲喂给药的最大无作用剂量为0.71mg·kg-1·d-1。若以100倍安全系数计,则其ADI为0.0071mg·kg-1·d-1。
With the speed development of industry, there are about 100 thousand chemicals in human daily life. It was proved that many kinds of chemicals could influence human reproductive function and make changes of human fertility particularly sperm count and quality. Peple had paid more and more attention to the damage of reproductive system by exotic chemicals.
Trichloroisocyanuric acid(TCCA) is a new, high-efficiency, wide-table, low-toxic, stable, effect-lasting, convenient disinfectant, which is already extensively applied in several industries(food profession,medical Industry,agriculture,fishery,textile and papermaking indu-stry). Although there have been some reports concerning on TCCA-induced toxicity of single species, few expriments on male reproductive toxicity and testicular injury were investigated.
This study here consists of three parts to determine the effect of TCCA on testicular strcture and function in SD rats, Exp.1 was to observe the long-term toxicity of TCCA in rats with various concentrations (0.71,2.29 and 7.59mg-kg-1·d-1) for 24 months.Exp.2 was to observe the short-term toxicity with the dosage of 96.5 and 193mg/kg through douche for 10 days and Exp,3 was to investigate the toxicity of TCCA and its molecular mechanisma with the dosage of 275 and 550 mg/kg through douche for 10 weeks (each other day)by the means of immunohistochemical method (SABC), real-time fluorescence quantitative PCR and atomic absorption spectrometry to detect the changes of Fas/FasL,Bcl-2/Bax-mediated apoptotic pathways, the contents of Zn,Ca and Cu in testis.
Main methods and results
1. Effect of TCCA on general conditions of Rats
A total of 270 rats,4 or 5 week-old, ingested TCCA in various concentrations (0.71,2.29,7.59mg·kg-1·d-1) for 24 months. And their general conditions, including behaviour, diet and drinking water had no significant difference compared with control group. When the rats were poisoned acutely through douche in the doses of 96.5,193mg/kg for 4 days, their physical activity decreased, back arched, and spirits sagged with no death in the experiment.When in the doses of 275,550mg/kg for 10 weeks,they immediately became sensitive to stimulus, too impatient and walked with the back arched.Then the rats had a dull reaction after 4 hours later and almost no activity after 5 weeks.Deaths happened with hemorrhagic secretions around oral and anal after 9 days.
These results suggested that the general conditions of rats exposed to TCCA varied with the dosage levels, meaning the higher the dosage of TCCA, the more obvious toxicity reactions, the early time of death.
2. Effect of TCCA on growth and reproductive organs weights of rats
In Exp.1, although the growth weights were not significantly lower in exposed rats than in control rats. the testicular weight loss occurred and organs index of testis decreased in exposed rats compared with the control rats. In Exp.2,the dosage of 193 mg/kg TCCA for 4 week-old rats could significantly decrease the weight and organs index of testis. And the dosage of 96.5 and 193mg/kg TCCA made the body weight loss both in 4 and 12 week-old rats. And in Exp.3, the body weight and testicular weight were also greatly decreased both in 275 and 550mg/kg groups.
All of the above demonstrated that the body weight of rats was significantly descend with dose increasing of TCCA,and exposed to TCCA long-time and low-dose or short-time and high-dose could cause weight loss of testis.
3. Effect of TCCA on Mean Seminiferous Tubules Dameter (MSTD) of testis
In this study, we observed the MSTD of testis with different concentration and operation time of TCCA.The results showed that the MSTD werel93.69,167.3,144.23,327.90 and 325.9μm correlated with dose of 7.59mg·kg-1·d-1,96.5 mg/kg in 4 week-old rat、193mg/kg in 4 week-old rat,96.5 and 193mg/kg in 12 week-old rat.All above MSDT were significantly declined compared with each controls and correlated with the dosage.These results suggested that TCCA could made seminiferous tubules thin and showed toxicity evidences of TCCA to testis.
4. Effect of TCCA on histomorphology of testis
The morphological changes of testis at 12 months characterized by atrophy of seminiferous tubules, arrest of spermatogenesis and smaller volume in 7.59mg-kg-1·d-1 goup rats,while there were no above-mentioned changes in control group. The incidence rates of above-mentioned changes at 24 month were significantly higher in 2.29 and 7.59 mg-kg-1·d-1 goup rats than that in control group. And the severity of histopathologic damage observed correlated positively with the increase of TCCA concentration and contact time. This histopathological examination suggested that TCCA could damag the structures of testis, cause disorder of spermatogenesis and make sperms disappear in epididymis. There was a obvious reproductive toxicity of TCCA and its primary target organ was testis in SD rat.
In the dose of 96.5 and 193mg/kg, the morphological changes of testis characterized by increased significantly degeneration of spermatogenic cells in 4 week-old rat,disorder and drop of spermatogenic cells, vacuoles in nucleus of Sertoli cells and degeneration of oblong spermatozoa in 12 week-old rat, the severity of histopathologic damage observed correlated positively with the increase of TCCA concentration
In the dose of 275 and 550mg/kg, the morphological changes of testis characterized by atrophy of seminiferous tubules, individual irregular tubular lumens, vacuoles in cytoplasm of Sertoli cells and noteworthy hyperplasia of testicular interstitial cells, and the severity of histopathologic damage observed correlated positively with the increase of TCCA concent-ration.
Additionally, in the dose of 193 mg/kg, the ultrastructure of spermatogonium was observed shown in detail as imperfect nuclear membrane, deformed nucleus, prominent clumped chromatin without clear apoptotic body and necrosis, while other cell organelles remained normal.
5. Effect of TCCA on expression of cell apoptosis associated ptoteins in testis of rats
In the study of toxic mechanisms induced by TCCA gavaged for 10 weeks (every other day), Immunohistochemical method was used to determine the expression of apoptosis associated proteins including Bcl-2,Bax,Fas,FasL,Caspase-3 and real-time fluorescenct quantitative PCR was uesed to carry on the quantitative analysis about gene expression of Bcl-2 and Fas mRNA.. The following results were obtained:
(1) Positive expression index of Bcl-2.Bax,Fas,FasL,Caspase-3 in control group and 275 mg/kg group were 67.97%/48.75%,58.99%/45.70%,82.31%/78.46%,53.79%/76.05% respectively, and there were significant differences between two groups except Fas.
(2) Bcl-2/Bax positive ratio in 275 mg/kg group were 1.06, which was significantly less than that in control group (1.15)
(3) There were higher positive intensity of Fas protein in 275 mg/kg group and significant difference in between two groups
(4) Compared with the control group, Bcl-2 and Fas genes were up-regulated in the 275 mg/kg group.
The results of this study suggest that apoptosis of spermatogenic cells can be induced by TCCA which may priming extrinsic and intrinsic pathways by up-regulating Fas/FasL and down-regulating Bcl-2/Bax proteins, in turn, activate the expression of downstream Caspase-3 proteins.
6. Effect of TCCA on the contents of Zn, Ca and Cu in testis of rats
We adopted atom absorption spectrophotometry technique to detect the contents of zinc (Zn), copper (Cu), and calcium (Ca) in testis and found that the contents of these three elements (Zn, Cu, Ca) in control,275and 550mg/kg groups were24.41/24.00/22.83 mg/kg, 2.03/1.99/1.59mg/kg,18.36/16.66/14.42 mg/kg respectively. The tendency of their contents was gradually decreasing with dose increasing, and in 550mg/kg group, there was significant difference compared with control group except the element of Ca.
7. Effect of TCCA on quality of rat's sperm.
We assessed sperm quality reduced by TCCA in a number of ways, for instance concentration, percentage of motile sperm and sperm shape.In the dose of 96.5 and 193 mg/ kg, the number of sperm was 428.5 and 420.9×106/g, and the percent of motile sperm was 75.6%and 64.84%, and there was no typical abnormal sperm observed. Compared to control group, both the number of sperm and the percent of motile sperm decreased with dose increasing,and there was significant difference between experimental and control groups. The final results confirmed that TCCA could significantly reduce quality of sperm.
8. Effect of TCCA on the Blood system of rats
After poisoned by TCCA for 6,12,18 and 24 months in the dosage of 0.71,2.29,7.59 mg·kg-1·d-1,the rat's blood were obtained to routine blood test and blood bio-chemical index examine. However there was no significant correlation between TCCA concentrations and every index of blood system. At the level of 195mg/kg for 10 days, the enzymatic activity of serum lactate dehydrogenase (LDH) activitie was increased and serum chloride, calcium level became lower. All of the above demonstrated that the effect of TCCA on blood system related to poisoned dosage. and the higher doses of TCCA administered, the more damage on blood system.
Conclusions
1. TCCA had obvious reproductive toxicity to testis and could cause pathological changes of testis with loss of testicular weight and atrophy of seminiferous tubules in the end.
2. The degree of testicular damage by TCCA proportionated to length of exposure and concentration. The higher doses of TCCA administered, the longer time of TCCA poisoned, and the more severity of damage on testis,
3. TCCA could significantly reduce the quality of sperm in rats,
4. As a reproductive toxic chemical, one of important toxic mechanism for TCCA was induced apoptosis of spermatogenic cells.
5. TCCA might make the reproductive toxicity to the testis. by means of reducing the contents of Zn and Cu,
6. The effect of TCCA on behaviour,body weight and blood system related to poisoned dosage.The maxial noneffective dose of TCCA was 0.71 mg-kg-1·d-1 in SD male rats and the acceptable daily intake(ADI) was 0.0071 mg-kg-1·d-1 with 100 times safety coefficient.
引文
[1]TCCA-生产无公害农产品之首选农药.农药市场信息,2003,(17)：27
[2]林雪梅TCCA的生产及应用.精细与专用化学品,2002,(21)：54-56.
[3]黄锦霞TCCA的新用途.氯碱工业,1987,(7)：32-33.
[4]余碧霞.杨晋云.夏顺利.广西化工.1990.(1)：40-43.
[5]邵明,杨文伟,葛敏.氯代异氰尿酸的性质及应用.氯碱工业,1999,8：27-29.
[6]王治顺,郑玉林,刘国良.高效消毒剂鱼安的主体有效成分-TCCA研制.氯碱工业.1988,(4)：38-39.
[7]任引津.实用急性中毒全书.北京：人民卫生出版社,2003
[8]黄斌英,王丽娟,刘华.1例TCCA中毒的急救护理体会.中国职业医学,2002,29(2)：43.
[9]王世俊：临床职业病学.北京：北京医科大学.中国协和医科大学联合出版,1994
[10]谢文菊,长玉莲,郭家祥.急性三氯异氰脲酸中毒事故调查.中华劳动卫生职业病杂志,1998,16(4)：232-233
[11]王朝阳,朱均.急性TCCA吸入性肺水肿1例.中国煤炭工业医学杂志,2006,9(10)：1098.
[12]房英春,李越,李胜男,等.二溴海因和TCCA对孔雀鱼的急性毒性试验.水产养殖,2005.26(6)：38-39.
[13]吴志新,陈孝煊,唐镇宇.红螫螯虾对3种药物耐受性的研究.华中农业大学学报,,2000.19(4)：381-383.
[14]徐镇,徐如卫,周志明等.四种常用渔药对秀丽白虾的急性毒性试验.水产科学,2005,24(7)：29-31.
[15]Kostich M S, Lazorchak J M,. Risks to aquatic organisms posed by human pharmaceu-ticaluse.Science of Total Environment,2008,389:329-339
[16]Bound J P,Voμlvoμlis N. Pharmaceuticals in the aquatic environment-a comparison of risk assessment strategies. Chemosphere,56(11):1143-1155
[17]Fent K, Weston A A, Caminada D.Ecotoxicology of human pharmaceuticals. Aquatic Toxicology,2006,76:122-159
[18]Costanzo SD, Murby J, Bates J. Ecosystem response to antibiotics entering theaquatic environment. Marine Pollution Bμlletin,2005,51(1-4):218-223
[19]章南岚,徐宁,段舜山TCCA对养殖海域浮游植物生长的影响.海洋环境科学,2009,28(1)：5-8
[20]聂湘平,王翔,李凯彬.TCCA对剑尾鱼(Xiphophorus helleri)毒性及其抗氧化酶影响.海洋与湖沼,2008,39(5)：494-497
[21]水生生物监测手册国家环保局《水生生物监测手册》编委会.南京：东南大学出版社,1993
[22]Nouguchi N, Nakada A, Itoh Y. Formation of active oxygen species and lipid peroxide induced by hypochlorite. Archive of Biochemistry& Biophysics,2002,397(2):440-445
[23]周孝治,陈菊芳,聂湘平,等TCCA在模拟水生态系统中归趋的研究.生态科学2007,26(4)：361-366
[24]郑艳艳,李羡筠,陈晓琴TCCA原药急性毒性和蓄积毒性试验.广西预防医学,2005,11(6)：355-357
[25]操璟璟,张小平,蒋继宏,等.农药TCCA对两种蚤类的慢性毒性,安徽师范大学学报：自然科学版,2007,30(4)：490-492
[26]李羡筠,陈晓琴,葛宪民,等.TCCA对大鼠的亚慢性毒性.中国工业医学杂志,2006,19(2)：103-105
[27]赵军,吕文清.三氯异氰脲酸的生产方法与应用.山西化工,2000,20(4)：51-52.
[28]周学永.三氯异氰脲酸的合成及应用开发.化学工程师,2000,77：36-37.
[29]苏彤.三氯异氰脲酸开发应用前景广阔.化工中间,2002,(8/9)：15-16.
[30]丁训诚,蒋学之,等.男性生殖毒理学.中国人口出版社.1997
[31]徐幼云,顾泽南译.饮水与健康//美国安全饮水委员会,饮水与健康.1977：424-425.
[32]Pryor JL,Hurt GS,Caloras D,et al. Histologic analysis of orchiopexy in a cryptorchid rabbit model. J Urol,1989,142:413-417.
[32]Martin D,Yves C.Role of spermatogonia in the repair of the seminiferous epithelium following X-irradiation of the rat testis.AM.J.Anat,128:265-282.
[33]Wing TY,Christensen AK. Morphometric studies on rat seminiferous tubμles.the American journal of anatomy,1982,165:13-25.
[34]Bansode FW,Dwivedi AK,Maikhuri JP,et al.Quantitative Analysis of Spermatogenesis in Rats Made Azoospermic With Compound CDRI 84/35. Contraception,1998,58:175- 82
[35]黄吉武,周宗灿.毒理学：毒物的基础科学.人民卫生出版社,2005
[36]徐晨,周作民.生殖生物学理论与实践.第一版.上海科学技术文献出版社,2005
[37]Smith RJF. Seasonal changes in the histology of the gonads and dorsal skin of the fathead minnow, Pimephales promelas.Can.J.Zool.,1978,56:2103-2109.
[38]Gimeno S,KomenH,Jobling S,etal.Demascμlinisation of Sexually mature male common carp,cyprinus carpio,exposed to 4-tertpentyl phenol during spermatogenesis.Aquat. Toxicol.1998,43:93-109
[39]Terranova,P.F.The Male Reproductive System(Chapter 37)in Medical Physiology,2003, 2nd edition, Lippincott, Williams&Wilkins (eds.R.Rhoades and G.Tanner)
[40]Vigier M,Weiss M,Perrard MH,et al.The effect s of FSH and of testosterone on the completion of meiosis and the very early steps of spermiogenesis of the rat:an in vitro study[J].J Mol Endocrinol,2004,33(3):729-742.
[41]Said TM, Paasch U,Glander HJ,et al.Role of caspases in male infertility. Hum Reprod Update,2004,10(1):392-351.
[42]Kilinc F, Guvel S, Kayaselcuk F, et al. p53 expression and poptosis in varicocele in the rattestis.J Urol,2004,172 (6 Pt 1):2475-2478.
[43]J Moretti E, Di Cairano G, Capitani S, et al. Cryptorchidism and semen quality:a TEM and molecular study.Andrology,2007,28(1):194-199.
[44]Assinder S, Davis R, Fenwick M, et al. Adμlt-only exposure of male rats to adiet of high phytoest rogen content increases apoptosis of meiotic and post-meiotic germ cells.Reproduction,2007,133(1):11-19.
[45]Said TM, Paasch U, Glander HJ, et al. Role of caspases in male infertility. Hum Reprod Update,2004,10(l):39-51.
[46]Sinha Hikim AP, Lue Y,Diaz2Romero M, et al. Deciphering the pathways of germ cell apoptosis in the testis.Jb Steroid Biochem Mol Biol,2003,85(225):175-182
[47]Theas MS,Rival C,Dietrich SJ,et al.Death recep tor and mi to chondrial pathways are involved in germ cell apop tosis in an experimental model of autoimmune orchitis. Hum Rep rod,2006,21(7):1734-1742.
[48]Embree-Ku M, Venturini D, Boekelheide K. Fas is involved in the p53-dependent apoptotic response to ionizing radiation in mouse testis Biol Reprod.2002,66 (5):1456-1461.
[49]Fujisawa M, Ishikawa T. Soluble forms of Fas and Fas ligand concent rations in t he seminal plasma of infertile men with varicocele. J Urol,2003,70(6 Pt 1):2363-2365.
[50]Hu JH,J iang J,Ma YH,et al. Enhancement of germ cell apoptosis induced by ethanol in transgenic mice overexp ressing Fas Ligand.Cell Res,2003,13(5):361-367.
[51]Ott M, Robertson JD,Gogvadze V,et al.Cytochrome crelease from mitochondria proceeds by a two-step process. Proc Natl Acad Sci USA,2002,99(3):1259-1263.
[52]Arnoμlt D.Apoptosis-associated mitochondrial outer membrane permeabilization assays.Methods,2008,4(3):229-234.
[53]Saikumar P,Dong Z,Weinberg J M,et al.Mechanisms of cell death in hypoxia/ reoxygenation injury. Oneogene,1998,7;3341-3347.
[54]Yang J,Liu X，Bhalla K,et al. Prevention of apoptosis by Bcl2:release of cytochrome C from mitochondria blocked. Science,1997,275:1129-1131.
[55]张明,袁慧.镉对小鼠生精细胞凋亡及Bax和Bcl-2基因表达的影响.中国兽医科学,2007,37(3)：251-254.
[56]林汝仙,孙志贤.Bcl-2基因家族与细胞程序性死亡.国外医学：遗传学分册,1997,20(5)：242.
[57]孙应彪,王丽娟,等.镍染毒大鼠睾丸生精细胞凋亡及其对Bcl-2和Bax表达的影响.毒理学杂志,2007,21(1)：36240.
[58]Korsm eyer S J,WeiM C,Saito M,et al Pro-apoptotic cascade activates BID, which oligomerizes BAK or BAX into pores that resμlt in the release of cytochromec.Cell Death Differ,2000,7(12):1166.
[59]Koji T, H ishikaw a Y. Germ cell apo pto sis and its molecular trigger in mouse testes. Arch Histoloytol,2003,66(1):1-16.
[60]Damavandi E, H ishikawa Y, Izumi S, et al. Involvement of Bax redist ribution in the induction of germcell apoptosis in neonatal mouse testes.Acta histochem cytochem, 2002,35(6):449-459.
[61]Billing H,Furuta I,Rivier C. Apoptosis in testis germ cells:developmental changes in gonadotropin dependence and localization to selective tubule stages. Endocrinology, 1995; 136,5-12
[62]Yin Y,DeWolf WC, Experimental cryptorchidism induces testicular germ cell apoptosis by p53-dependent and-independent pathways in mice.Morgentalor A,Biol Reprod, 1998; 58:492-496
[63]Sinha Hikim AP,Lue Y, Diaz-Romero M, et al. Deci phering the Pathways of germ cell apoptosisi in the testis J Steroid Biochem Mol Biol,2003,85(2-5):175-182.
[64]Suominen JS,Linderborg J,NiKμla H,et al.The effects of mono-2-ethylhexyl phathala-te adriamycin and N-ethyl-N-nitrosourea on stage-specific apoptosis andDNA synthesis in the mouse spermatogenesis.Toxicol Lett,2003,143(2):163-173
[65]Homma-Takeda S,Kugenuma Y,lwanuro T,er al. Impairment of spermatogenesis in rats by methylmercury:involvement of stageand cell-specific germ cell apoptosis. Toxieolo-gy,2001,169(1):25-35.
[66]Lee J,Richburg,JH,et al.The Fas system is a key regulator ofgerm cell apoptosis in the testis.Endocrinology,1997,138(5):2081-2088.
[67]Ichimura T,Kawamura M,Mitani A. Co-localized expression of FasL,Fas,Caspase 3 and apoptotic DNA fragmentation in mouse testis after oral exposure to di(2-ethylhe-xyl) phthalate. Toxicology,2003,194(1-2):35-42.
[68]Koji T,Hishikawa Y, Ando H, Nakanishi Y, Kobayashi N. Expression of Fas and Fas ligand in normal and ischemia-reperfusion testes:involvement of the Fas system in the induction of germ cell apoptosis in the damaged mouse testis.Biol Reprod,2001,64 (3):946-954.
[69]D'Abrizio P,Baldini E,Russop F,et al.ontogenesis and cell specific loealization of Fas ligand expression in the rat testis.Int J Androl,2004,27(5):304-310.
[70]Hunt CD,Johnson PE,Herbel J,et al.Effects of dietary zinc depletion on seminal volume and zinc loss, serum tegtosterone concentrations, and sperm morphology in young man. Am J ClinNutr,1992,56:148-157.
[71]Taylor MF, de Boer-Brouwer M, Woolveridge l,et al.Leydig cell apoptosis after administration of echane dimethanesμlfonate to the adμlt male rat is a Fas-mediated process. Endocrinology,1999,140(8):3797-3804
[72]翟中和,王喜忠,丁明孝.细胞生物学.北京：高等教育出版社,2000
[73]Rodriguez I, Ody C, Araki K, et al. An early massive wave of germinal cell apoptosis is required for the development of functional spermat ogenesis.The EMBO Journal, 1997,16(9):2262-2270.
[75]Blanco Rodriguez J. A matter of death and life:the significance of germ cell death during spermatogenesis. Int J Androl,1998,21 (5):236-248.
[76]Sinha Hikim A P, Swerdloff R S. Hormonal and genetic control of germ cell apoptosis in the testis. RevReprod,1999,4(1):38-47.
[77]Heninger N L, Staub C, Blanchard T L, et al. Germ cell apoptosis in the testes of normal stallions. Theriogenology,2004,62(1-2):283-297.
[78]Sasso Cerri E,Cerri PS,Fr eymμller E,et al.Apoptosis during the seasonal spermato-genic cycle of Rana cates beina. J Anat,2006,209(1):21-29
[79]Zheng S, T ur ner T T, Lysiak J J. Caspase 2 activity contributes to the initial wave of germ cell a poptosis during the first round of spermatog enesis.Biol Reprod,2006,74 (6):1026-1033.
[80]Vera Y, Rodriguez S, Castanares M, et al. Functional role of caspases in heated induced testicμlar germ cell apoptosis.Biol reprod,2005,72(3):516-522
[81]Moreno R D, Lizama C, Ur zua N, et al. Caspase activation throughout the first wave of spermatog enesis in the rat. Cell Tissue Res,,2006,325(3):533-540.
[82]Abe H,Shibata MA,Otsuki Y. Caspase cascade of Fas-mediated apoptosis in human normal endometrium and endometrial carcinoma cells.Mol Hum Reprod,2006,12 (9)535-541.
[83]Bucci B, D'Agnano ,I Amendola D, et al. Myc down-regμlation, sensitizes melanoma cells to radiotherapy by inhibiting MLH1 and MSH2 mismatch repair proteins.Clin Cancer Res,2005,11(7):2756-2767.
[84]Chan S,Gerson B,Sub ramaniam S. The Role of Copper, Molybdenum, Selenium, and Zinc in Nutrition and Health.Clin Lab Med,1998,18 (4):673-685.
[85]Oteiza PI.Zinc deficiency causes oxidative damage to proteins,lipids and DNA in rat testes.J Nutr,1995,125:823-829.
[86]Matsushita K,Kitagawa K,Matsuyama T,et al.Effect of systemic zinc administration on delayed neuronal death in the gerbil hippocampus. Brain Res,1996,743 (1-2):362-365.
[87]Koizumi T,Waalkes MP.Effects of zinc on the distribution and toxicy of cadmium in isolated interstitial cells of the rat testis.Toxicollgy,1989,56:137-146
[88]Carlsen E,Giwercman A,Keiding N,etal.Evidence for decreasing quality of semen during past 50 years.Br Med J,1992;305:609-613
[89]张树成,王弘毅,王介东.1981-1996年我国有生育力男性精液质量的变化分析.生殖与避孕,1999,19(1)：27-53
[1]Kerr JF,Wyllie AH,Currie AR.Apoptosis:a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer,1972,26(4):239-257.
[2]John Savill. Apoptosis:A mechanism for regulation of the cell complement of inflamed glole.Ruli.Kidned.lnt,1992,41:607.
[3]Kilinc F, Guvel S,Kayaselcuk F,et al. p53 expression and apoptosis in varicocele in the rat testis. J Urol,2004,172(6 pt 1):2475-2478.
[4]Martin D,Yves C.role of spermatogonia in the repair of the seminiferous epithelium following X-irradiation of the rat testis.Am.J.Anat,128:265-282.
[5]Billing AD. Apoptosis in the testis germ cells:developmental changes in Gonadotr-opindependence and localization to selective tubule stages. Endocrinology,1995,136, (2): 15-17.
[6]Beaulaton J,Lockshin RA. The relation of programmed cell death to development and reproduction:comparative studies and an attempt at classification. Int. Rev. Cytol,1982,79 (2):215.
[7]Kerr JB. Spontaneous degeneration of germ cells in normal rat testis:assessment of cell types and frequency during the spermatogenic cycle.J Reprod Fertil,1992,95 (3):825-830.
[8]Mori C. Morphological analysis of germ cells during postnatal testis development in normal and HSP70-2 knockout mice. Dev Dyn,1997,208:125-126.
[9]Blanco-Rodriguez,J.and Martinez-Garcia,C. Spontaneous germ cell death in the testis of the adult rat takes the form of apoptosis:re-evaluation of cell types that exhibit the ability to die during spermatogenesis. Cell.Prolif.,29:13-31.
[10]Eddy EM. Male germ cell gene expression. Recent Prog Horm Res,2002,57:103-128.
[11]Theas MS,Rival C,Dietrich SJ,et al. Death receptor and mitochondrial pathways are involvedigerm cell apoptosis in an experimental model of autoimmune or chitis. Hum Reprod,2006,21(7):734-1742.
[12]Assinder S,Davis R,Fenwick M,et al. Adult-only exposure of male rats to a diet of high phytoestrogen content increases apoptosis of meiotic and post-meiotic germ cells. Reproduction,2007,133(1):11-19.
[13]曹兴午,王传航,周强,等.不育症精液生精细胞凋亡率检测.生殖医学杂志,2008,17(5)：351-355.
[14]Kinner MK,Fetterolf PM,Anthony CT. Purification of aparacrine factor, P-Mod-S, produeed by testicular peritubular cells that modulates Sertoli cell function.JBiol Chem.1988.263(6):2884-2890.
[15]Anthony CT,Rosselli M,Skinner MK.Aetions of the testicular paracrine factor (P-Mod-S) on Sertoli cell transferring seeretion throughout pubertal development. Endocrinology. 1991,129(1):353-360.
[16]Sousa M,Cremades N,Alves C,et al. Developmental potential of human spermatogenic cells co-cultured with Sertoli cells. Hum Reprod,2002,17 (1):161-172.
[17]Lacham-Kaplan O. In vivo and in vitro differentiation of male germ cells in the mouse. Reproduction,2004,128:147-152.
[18]Rooij DG. Proliferation and differentiation of spermatogonial stem cells.Reproduction, 2001,121:347-354.
[19]王心如,周宗灿.2003.毒理学基础(第4版).北京：人民卫生出版社.224-238.
[20]Meroni S,Canepa D,Pellizzari E,et al. Regulation of gammaglutamyl transpeptidase activity by Ca2+and protein kinase C-de-pendent pathways in Sertoli cells. Int J Androl, 1997,20(4):189-194.
[21]Vigier M, Weiss M. Perrard MH, et al. The effects of FSH and of testosterone on the comp letion of meiosis and the very early steps of spermiogenesis of the rat:an in vitro study [J]. J Mol Endocrinol,2004,33 (3):729-742.
[22]Woolveridge I,de Boer-Brouwer M,Taylor MF, et al. Apoptosis in the rat spermatogenic epithelium following androgen withdrawal:changes in apoptosis-related genes. Biol Reprod,1999,60 (2):461-470.
[23]Hikim,A.P. Involvement of apoptosis in the induction of germ cell degeneration in adult rats after gonadotropin-releasing hormone antagonist treatment. Endocrinology,1995. 136(6):2770-2775.
[24]Hikim P P,Wang C,Leung A,et al. Involvement of apoptosis in the induction of germ cell degeneration in adult rats after gonadotropin releasing hormone antagonist treatment. Endocrinology,1995,136,(6)1:2770.
[25]Lei Z M, Mishra S, Ponnuru P, et al. Testicular phenotype in luteinizing hormone recep tor knockout animals and the effect of testosterone replacement therapy. Biol Rep rod 2004,71 (5):1605-1613.
[26]崔毓桂,陈子庆,沙家豪.激素对精子发生的调控.中华男科学,2004,10(6)：465-467.
[27]Sinha Hikim AP,Lue Y,Diaz-Romero M,et al. DeciPhering the palthways of germ cell apoptosis [J].J Steroid Biochem Mol Biol,2003,85(2-5):175-182.
[28]Bakalska,M. Induction of male germ cell apoptosis by testosterone withdrawal after ethane dimethanesulfonate treatment in adult rats. Endocr Regul,2004.38 (3):103-110.
[29]Tesarik J,M.endoza C,Greco E. The effect of FSH on male germ cell survival and differentiation in vitro is mimicked by pentoxifylline but not insulin. Mol Hum Reprod, 2000,6(10):877-881.
[30]Nandi S,Banerjee PP,Zirkin BR. Germ cell apoptosis in the testes of Sprague-Dawley rats following testosterone withdrawal by ethane1,2-dimethane-sulfonate administ ration relationship to Fas. Biol Reprod,1999,61 (1):70-75.
[31]Mishra,D.P.and C.Shaha,Estrogen-induced spermatogenic cell apoptosis occurs via the mitochondrial pathway:role of superoxide and nitric oxide. J Biol Chem,2005.280 (7): 6181-6196.
[32]Yazawa,H.,I. Sasagawa,T.Nakada,Apoptosis of testicular germ cells induced by exogen-ous glucocorticoid in rats.Hum Reprod,2000.15(9):1917-1920.
[33]Koji T. Male germ cell death in mouse testes:possible involvement of Fas and Fas ligand. Med Electron Microsc,2001,34(4):213-22.
[34]Bhardwaj A&Aggarwal BB. Receptor-mediated choreography of life and death. J Clin Immunol,2003,23(5):317-32.
[35]Philchenkov AA. Caspases as regulators of apoptosis and other cell functions. Biochemi-stry (Mosc),2003,68(4):365-376.
[36]李宏军,俞莉章,郭应禄,等.Fas在正常睾丸组织及精原细胞瘤中的表达.中华男科学,2001,7(3)：150-153.
[37]Mizutani Y, Yoshida O, Bonavida B. Prognostic significance of soluble Fas in the serum of patients with bladder cancer. J Urol,1998,160 (2):571-576.
[38]Eguchi J,Koji T,Nomata K,et al. Fas-Fas ligand system as a possible mediator of sperma-togenic cell apoptosis in human maturation-arreseted testes.Hum Cell,2002,15(1): 61-68.
[39]Lee J, Richburg J H, Shipp EB, et al. The Fas system,a regulator of testicular germ cell apoptosis, is differentially up-regulated in Sertoli cell versus germ cell injury of the testis. Endocrinology,1999,140 (2):852-858.
[40]Richburg J H,Nanez A, Gao H. Participation of the Fas-signaling system in the initiation of germ cell apoptosis in young rat testes after exposure to mono-(2-ethylhe-xyl) phthalate. Toxicol Appl Pharmacol,1999,160 (3):271-278.
[41]Fujisawa M, Ishikawa T. Soluble forms of Fas and Fas ligand concentrations in the seminal plas ma of infertile men with varicocele. J Urol,2003,170(6Pt1):2363-2365.
[42]MacFarlane M,Williams C. Apoptosis and disease:a life or death decision.nat ure. EMBO reports,2004,5(7):674-678.
[43]Yamamoto CM,Sinha Hikim AP,Huynh PN,et al. Redistribution of Bax is an early step in an apoptotic pathway leading to germ cell death in rats,triggered by mild testicular hypothermia. Biol Reprod,2000,63(6):1683-1690.
[44]Oldereid NB,Angelis PD,Wiger R,et al. Expression of Bcl-2 family proteins and spontaneous apoptosis in normal human testis. Mol Hum Reprod,2001,7(5):403-8.
[45]Zhang ZH,Jin X.Zhang XS,et al. Bcl-2 and Bax are involved in experimental cryptorchidism-induced testicular germ cell apoptosis in rhesus monkey. Contraception, 2003,68(4):297-301.
[46]Eskes R,Desagher S,Antonsson B,et al. Bid induces the oligomerization and insertion of Bax into outer mitochondrial membrane. Mol Cell Biol,2000,20(3):929-935.
[47]Chandrasekaran,Y,J.H.Richburg. The p53 protein influences the sensitivity of testicular germ cells to mono-(2-ethylhexyl)phthalate-induced apoptosis by increasing the membrane levels of Fas and DR5 and decreasing the intracellular amount of c-FLIP.Biol Reprod,2005.72(1):206-13.
[48]Wunderlich M&Berberich SJ. Mdm2 inhibition of p53 induces E2F1 transactivation via p21.Oncogene,2002,21(28):4414-4421.
[49]Yin Y,DeWolf WC, Experimental cryptorchidism induces testicular germ cell apoptosis by p53-dependent and-independent pathways in mice. Morgentalor A,Biol Reprod,1998; 58:492-496
[50]Kilinc F,Guvel S,Kayaselcuk F,et al. p53 expression and apoptosis in varicocele in the rat tesis. J Urol,2004,172 (6 Ptl):2475-2478.
[51]Gao CF,Ren S,Zhang L,et al. Caspase-dependent cytosolic release of cytochrome c and membrane translocation of Bax in p53-induced apoptosis. Exp Cell Res,2001,265.(1): 145-151.
[52]Singh NP,Muller CH&Berger RE. Effects of age on DNA double-strand breaks and apoptosis in human sperm. Fertil Steril,2003,80(6):1420-1430.
[53]Erenpreisa J,Erenpreiss J,Freivalds T,et al. Toluidine blue test for sperm DNA integrity and elaboration of image cytometry algorithm. Cytometry,2003,52 (1):19-27.
[54]Jersmann HP,Ross KA,Vivers S,et al. Phagocytosis of apoptotic cells by human macroph-ages:analysis by multi-parameter flow cytometry. Cytometry,2003,51 (1):7-15.
[55]林本成,袭著革,张英鸽,等.应用流式细胞分析技术研究微纳尺度Si 0：对雄性大鼠睾丸生精细胞的毒性作用.生态毒理学报,2007.2(3)：322-326
[2]林雪梅TCCA的生产及应用.精细与专用化学品,2002,(21)：54-56.
[3]黄锦霞TCCA的新用途.氯碱工业,1987,(7)：32-33.
[4]余碧霞.杨晋云.夏顺利.广西化工.1990.(1)：40-43.
[5]邵明,杨文伟,葛敏.氯代异氰尿酸的性质及应用.氯碱工业,1999,8：27-29.
[6]王治顺,郑玉林,刘国良.高效消毒剂鱼安的主体有效成分-TCCA研制.氯碱工业.1988,(4)：38-39.
[7]任引津.实用急性中毒全书.北京：人民卫生出版社,2003
[8]黄斌英,王丽娟,刘华.1例TCCA中毒的急救护理体会.中国职业医学,2002,29(2)：43.
[9]王世俊：临床职业病学.北京：北京医科大学.中国协和医科大学联合出版,1994
[10]谢文菊,长玉莲,郭家祥.急性三氯异氰脲酸中毒事故调查.中华劳动卫生职业病杂志,1998,16(4)：232-233
[11]王朝阳,朱均.急性TCCA吸入性肺水肿1例.中国煤炭工业医学杂志,2006,9(10)：1098.
[12]房英春,李越,李胜男,等.二溴海因和TCCA对孔雀鱼的急性毒性试验.水产养殖,2005.26(6)：38-39.
[13]吴志新,陈孝煊,唐镇宇.红螫螯虾对3种药物耐受性的研究.华中农业大学学报,,2000.19(4)：381-383.
[14]徐镇,徐如卫,周志明等.四种常用渔药对秀丽白虾的急性毒性试验.水产科学,2005,24(7)：29-31.
[15]Kostich M S, Lazorchak J M,. Risks to aquatic organisms posed by human pharmaceu-ticaluse.Science of Total Environment,2008,389:329-339
[16]Bound J P,Voμlvoμlis N. Pharmaceuticals in the aquatic environment-a comparison of risk assessment strategies. Chemosphere,56(11):1143-1155
[17]Fent K, Weston A A, Caminada D.Ecotoxicology of human pharmaceuticals. Aquatic Toxicology,2006,76:122-159
[18]Costanzo SD, Murby J, Bates J. Ecosystem response to antibiotics entering theaquatic environment. Marine Pollution Bμlletin,2005,51(1-4):218-223
[19]章南岚,徐宁,段舜山TCCA对养殖海域浮游植物生长的影响.海洋环境科学,2009,28(1)：5-8
[20]聂湘平,王翔,李凯彬.TCCA对剑尾鱼(Xiphophorus helleri)毒性及其抗氧化酶影响.海洋与湖沼,2008,39(5)：494-497
[21]水生生物监测手册国家环保局《水生生物监测手册》编委会.南京：东南大学出版社,1993
[22]Nouguchi N, Nakada A, Itoh Y. Formation of active oxygen species and lipid peroxide induced by hypochlorite. Archive of Biochemistry& Biophysics,2002,397(2):440-445
[23]周孝治,陈菊芳,聂湘平,等TCCA在模拟水生态系统中归趋的研究.生态科学2007,26(4)：361-366
[24]郑艳艳,李羡筠,陈晓琴TCCA原药急性毒性和蓄积毒性试验.广西预防医学,2005,11(6)：355-357
[25]操璟璟,张小平,蒋继宏,等.农药TCCA对两种蚤类的慢性毒性,安徽师范大学学报：自然科学版,2007,30(4)：490-492
[26]李羡筠,陈晓琴,葛宪民,等.TCCA对大鼠的亚慢性毒性.中国工业医学杂志,2006,19(2)：103-105
[27]赵军,吕文清.三氯异氰脲酸的生产方法与应用.山西化工,2000,20(4)：51-52.
[28]周学永.三氯异氰脲酸的合成及应用开发.化学工程师,2000,77：36-37.
[29]苏彤.三氯异氰脲酸开发应用前景广阔.化工中间,2002,(8/9)：15-16.
[30]丁训诚,蒋学之,等.男性生殖毒理学.中国人口出版社.1997
[31]徐幼云,顾泽南译.饮水与健康//美国安全饮水委员会,饮水与健康.1977：424-425.
[32]Pryor JL,Hurt GS,Caloras D,et al. Histologic analysis of orchiopexy in a cryptorchid rabbit model. J Urol,1989,142:413-417.
[32]Martin D,Yves C.Role of spermatogonia in the repair of the seminiferous epithelium following X-irradiation of the rat testis.AM.J.Anat,128:265-282.
[33]Wing TY,Christensen AK. Morphometric studies on rat seminiferous tubμles.the American journal of anatomy,1982,165:13-25.
[34]Bansode FW,Dwivedi AK,Maikhuri JP,et al.Quantitative Analysis of Spermatogenesis in Rats Made Azoospermic With Compound CDRI 84/35. Contraception,1998,58:175- 82
[35]黄吉武,周宗灿.毒理学：毒物的基础科学.人民卫生出版社,2005
[36]徐晨,周作民.生殖生物学理论与实践.第一版.上海科学技术文献出版社,2005
[37]Smith RJF. Seasonal changes in the histology of the gonads and dorsal skin of the fathead minnow, Pimephales promelas.Can.J.Zool.,1978,56:2103-2109.
[38]Gimeno S,KomenH,Jobling S,etal.Demascμlinisation of Sexually mature male common carp,cyprinus carpio,exposed to 4-tertpentyl phenol during spermatogenesis.Aquat. Toxicol.1998,43:93-109
[39]Terranova,P.F.The Male Reproductive System(Chapter 37)in Medical Physiology,2003, 2nd edition, Lippincott, Williams&Wilkins (eds.R.Rhoades and G.Tanner)
[40]Vigier M,Weiss M,Perrard MH,et al.The effect s of FSH and of testosterone on the completion of meiosis and the very early steps of spermiogenesis of the rat:an in vitro study[J].J Mol Endocrinol,2004,33(3):729-742.
[41]Said TM, Paasch U,Glander HJ,et al.Role of caspases in male infertility. Hum Reprod Update,2004,10(1):392-351.
[42]Kilinc F, Guvel S, Kayaselcuk F, et al. p53 expression and poptosis in varicocele in the rattestis.J Urol,2004,172 (6 Pt 1):2475-2478.
[43]J Moretti E, Di Cairano G, Capitani S, et al. Cryptorchidism and semen quality:a TEM and molecular study.Andrology,2007,28(1):194-199.
[44]Assinder S, Davis R, Fenwick M, et al. Adμlt-only exposure of male rats to adiet of high phytoest rogen content increases apoptosis of meiotic and post-meiotic germ cells.Reproduction,2007,133(1):11-19.
[45]Said TM, Paasch U, Glander HJ, et al. Role of caspases in male infertility. Hum Reprod Update,2004,10(l):39-51.
[46]Sinha Hikim AP, Lue Y,Diaz2Romero M, et al. Deciphering the pathways of germ cell apoptosis in the testis.Jb Steroid Biochem Mol Biol,2003,85(225):175-182
[47]Theas MS,Rival C,Dietrich SJ,et al.Death recep tor and mi to chondrial pathways are involved in germ cell apop tosis in an experimental model of autoimmune orchitis. Hum Rep rod,2006,21(7):1734-1742.
[48]Embree-Ku M, Venturini D, Boekelheide K. Fas is involved in the p53-dependent apoptotic response to ionizing radiation in mouse testis Biol Reprod.2002,66 (5):1456-1461.
[49]Fujisawa M, Ishikawa T. Soluble forms of Fas and Fas ligand concent rations in t he seminal plasma of infertile men with varicocele. J Urol,2003,70(6 Pt 1):2363-2365.
[50]Hu JH,J iang J,Ma YH,et al. Enhancement of germ cell apoptosis induced by ethanol in transgenic mice overexp ressing Fas Ligand.Cell Res,2003,13(5):361-367.
[51]Ott M, Robertson JD,Gogvadze V,et al.Cytochrome crelease from mitochondria proceeds by a two-step process. Proc Natl Acad Sci USA,2002,99(3):1259-1263.
[52]Arnoμlt D.Apoptosis-associated mitochondrial outer membrane permeabilization assays.Methods,2008,4(3):229-234.
[53]Saikumar P,Dong Z,Weinberg J M,et al.Mechanisms of cell death in hypoxia/ reoxygenation injury. Oneogene,1998,7;3341-3347.
[54]Yang J,Liu X，Bhalla K,et al. Prevention of apoptosis by Bcl2:release of cytochrome C from mitochondria blocked. Science,1997,275:1129-1131.
[55]张明,袁慧.镉对小鼠生精细胞凋亡及Bax和Bcl-2基因表达的影响.中国兽医科学,2007,37(3)：251-254.
[56]林汝仙,孙志贤.Bcl-2基因家族与细胞程序性死亡.国外医学：遗传学分册,1997,20(5)：242.
[57]孙应彪,王丽娟,等.镍染毒大鼠睾丸生精细胞凋亡及其对Bcl-2和Bax表达的影响.毒理学杂志,2007,21(1)：36240.
[58]Korsm eyer S J,WeiM C,Saito M,et al Pro-apoptotic cascade activates BID, which oligomerizes BAK or BAX into pores that resμlt in the release of cytochromec.Cell Death Differ,2000,7(12):1166.
[59]Koji T, H ishikaw a Y. Germ cell apo pto sis and its molecular trigger in mouse testes. Arch Histoloytol,2003,66(1):1-16.
[60]Damavandi E, H ishikawa Y, Izumi S, et al. Involvement of Bax redist ribution in the induction of germcell apoptosis in neonatal mouse testes.Acta histochem cytochem, 2002,35(6):449-459.
[61]Billing H,Furuta I,Rivier C. Apoptosis in testis germ cells:developmental changes in gonadotropin dependence and localization to selective tubule stages. Endocrinology, 1995; 136,5-12
[62]Yin Y,DeWolf WC, Experimental cryptorchidism induces testicular germ cell apoptosis by p53-dependent and-independent pathways in mice.Morgentalor A,Biol Reprod, 1998; 58:492-496
[63]Sinha Hikim AP,Lue Y, Diaz-Romero M, et al. Deci phering the Pathways of germ cell apoptosisi in the testis J Steroid Biochem Mol Biol,2003,85(2-5):175-182.
[64]Suominen JS,Linderborg J,NiKμla H,et al.The effects of mono-2-ethylhexyl phathala-te adriamycin and N-ethyl-N-nitrosourea on stage-specific apoptosis andDNA synthesis in the mouse spermatogenesis.Toxicol Lett,2003,143(2):163-173
[65]Homma-Takeda S,Kugenuma Y,lwanuro T,er al. Impairment of spermatogenesis in rats by methylmercury:involvement of stageand cell-specific germ cell apoptosis. Toxieolo-gy,2001,169(1):25-35.
[66]Lee J,Richburg,JH,et al.The Fas system is a key regulator ofgerm cell apoptosis in the testis.Endocrinology,1997,138(5):2081-2088.
[67]Ichimura T,Kawamura M,Mitani A. Co-localized expression of FasL,Fas,Caspase 3 and apoptotic DNA fragmentation in mouse testis after oral exposure to di(2-ethylhe-xyl) phthalate. Toxicology,2003,194(1-2):35-42.
[68]Koji T,Hishikawa Y, Ando H, Nakanishi Y, Kobayashi N. Expression of Fas and Fas ligand in normal and ischemia-reperfusion testes:involvement of the Fas system in the induction of germ cell apoptosis in the damaged mouse testis.Biol Reprod,2001,64 (3):946-954.
[69]D'Abrizio P,Baldini E,Russop F,et al.ontogenesis and cell specific loealization of Fas ligand expression in the rat testis.Int J Androl,2004,27(5):304-310.
[70]Hunt CD,Johnson PE,Herbel J,et al.Effects of dietary zinc depletion on seminal volume and zinc loss, serum tegtosterone concentrations, and sperm morphology in young man. Am J ClinNutr,1992,56:148-157.
[71]Taylor MF, de Boer-Brouwer M, Woolveridge l,et al.Leydig cell apoptosis after administration of echane dimethanesμlfonate to the adμlt male rat is a Fas-mediated process. Endocrinology,1999,140(8):3797-3804
[72]翟中和,王喜忠,丁明孝.细胞生物学.北京：高等教育出版社,2000
[73]Rodriguez I, Ody C, Araki K, et al. An early massive wave of germinal cell apoptosis is required for the development of functional spermat ogenesis.The EMBO Journal, 1997,16(9):2262-2270.
[75]Blanco Rodriguez J. A matter of death and life:the significance of germ cell death during spermatogenesis. Int J Androl,1998,21 (5):236-248.
[76]Sinha Hikim A P, Swerdloff R S. Hormonal and genetic control of germ cell apoptosis in the testis. RevReprod,1999,4(1):38-47.
[77]Heninger N L, Staub C, Blanchard T L, et al. Germ cell apoptosis in the testes of normal stallions. Theriogenology,2004,62(1-2):283-297.
[78]Sasso Cerri E,Cerri PS,Fr eymμller E,et al.Apoptosis during the seasonal spermato-genic cycle of Rana cates beina. J Anat,2006,209(1):21-29
[79]Zheng S, T ur ner T T, Lysiak J J. Caspase 2 activity contributes to the initial wave of germ cell a poptosis during the first round of spermatog enesis.Biol Reprod,2006,74 (6):1026-1033.
[80]Vera Y, Rodriguez S, Castanares M, et al. Functional role of caspases in heated induced testicμlar germ cell apoptosis.Biol reprod,2005,72(3):516-522
[81]Moreno R D, Lizama C, Ur zua N, et al. Caspase activation throughout the first wave of spermatog enesis in the rat. Cell Tissue Res,,2006,325(3):533-540.
[82]Abe H,Shibata MA,Otsuki Y. Caspase cascade of Fas-mediated apoptosis in human normal endometrium and endometrial carcinoma cells.Mol Hum Reprod,2006,12 (9)535-541.
[83]Bucci B, D'Agnano ,I Amendola D, et al. Myc down-regμlation, sensitizes melanoma cells to radiotherapy by inhibiting MLH1 and MSH2 mismatch repair proteins.Clin Cancer Res,2005,11(7):2756-2767.
[84]Chan S,Gerson B,Sub ramaniam S. The Role of Copper, Molybdenum, Selenium, and Zinc in Nutrition and Health.Clin Lab Med,1998,18 (4):673-685.
[85]Oteiza PI.Zinc deficiency causes oxidative damage to proteins,lipids and DNA in rat testes.J Nutr,1995,125:823-829.
[86]Matsushita K,Kitagawa K,Matsuyama T,et al.Effect of systemic zinc administration on delayed neuronal death in the gerbil hippocampus. Brain Res,1996,743 (1-2):362-365.
[87]Koizumi T,Waalkes MP.Effects of zinc on the distribution and toxicy of cadmium in isolated interstitial cells of the rat testis.Toxicollgy,1989,56:137-146
[88]Carlsen E,Giwercman A,Keiding N,etal.Evidence for decreasing quality of semen during past 50 years.Br Med J,1992;305:609-613
[89]张树成,王弘毅,王介东.1981-1996年我国有生育力男性精液质量的变化分析.生殖与避孕,1999,19(1)：27-53
[1]Kerr JF,Wyllie AH,Currie AR.Apoptosis:a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer,1972,26(4):239-257.
[2]John Savill. Apoptosis:A mechanism for regulation of the cell complement of inflamed glole.Ruli.Kidned.lnt,1992,41:607.
[3]Kilinc F, Guvel S,Kayaselcuk F,et al. p53 expression and apoptosis in varicocele in the rat testis. J Urol,2004,172(6 pt 1):2475-2478.
[4]Martin D,Yves C.role of spermatogonia in the repair of the seminiferous epithelium following X-irradiation of the rat testis.Am.J.Anat,128:265-282.
[5]Billing AD. Apoptosis in the testis germ cells:developmental changes in Gonadotr-opindependence and localization to selective tubule stages. Endocrinology,1995,136, (2): 15-17.
[6]Beaulaton J,Lockshin RA. The relation of programmed cell death to development and reproduction:comparative studies and an attempt at classification. Int. Rev. Cytol,1982,79 (2):215.
[7]Kerr JB. Spontaneous degeneration of germ cells in normal rat testis:assessment of cell types and frequency during the spermatogenic cycle.J Reprod Fertil,1992,95 (3):825-830.
[8]Mori C. Morphological analysis of germ cells during postnatal testis development in normal and HSP70-2 knockout mice. Dev Dyn,1997,208:125-126.
[9]Blanco-Rodriguez,J.and Martinez-Garcia,C. Spontaneous germ cell death in the testis of the adult rat takes the form of apoptosis:re-evaluation of cell types that exhibit the ability to die during spermatogenesis. Cell.Prolif.,29:13-31.
[10]Eddy EM. Male germ cell gene expression. Recent Prog Horm Res,2002,57:103-128.
[11]Theas MS,Rival C,Dietrich SJ,et al. Death receptor and mitochondrial pathways are involvedigerm cell apoptosis in an experimental model of autoimmune or chitis. Hum Reprod,2006,21(7):734-1742.
[12]Assinder S,Davis R,Fenwick M,et al. Adult-only exposure of male rats to a diet of high phytoestrogen content increases apoptosis of meiotic and post-meiotic germ cells. Reproduction,2007,133(1):11-19.
[13]曹兴午,王传航,周强,等.不育症精液生精细胞凋亡率检测.生殖医学杂志,2008,17(5)：351-355.
[14]Kinner MK,Fetterolf PM,Anthony CT. Purification of aparacrine factor, P-Mod-S, produeed by testicular peritubular cells that modulates Sertoli cell function.JBiol Chem.1988.263(6):2884-2890.
[15]Anthony CT,Rosselli M,Skinner MK.Aetions of the testicular paracrine factor (P-Mod-S) on Sertoli cell transferring seeretion throughout pubertal development. Endocrinology. 1991,129(1):353-360.
[16]Sousa M,Cremades N,Alves C,et al. Developmental potential of human spermatogenic cells co-cultured with Sertoli cells. Hum Reprod,2002,17 (1):161-172.
[17]Lacham-Kaplan O. In vivo and in vitro differentiation of male germ cells in the mouse. Reproduction,2004,128:147-152.
[18]Rooij DG. Proliferation and differentiation of spermatogonial stem cells.Reproduction, 2001,121:347-354.
[19]王心如,周宗灿.2003.毒理学基础(第4版).北京：人民卫生出版社.224-238.
[20]Meroni S,Canepa D,Pellizzari E,et al. Regulation of gammaglutamyl transpeptidase activity by Ca2+and protein kinase C-de-pendent pathways in Sertoli cells. Int J Androl, 1997,20(4):189-194.
[21]Vigier M, Weiss M. Perrard MH, et al. The effects of FSH and of testosterone on the comp letion of meiosis and the very early steps of spermiogenesis of the rat:an in vitro study [J]. J Mol Endocrinol,2004,33 (3):729-742.
[22]Woolveridge I,de Boer-Brouwer M,Taylor MF, et al. Apoptosis in the rat spermatogenic epithelium following androgen withdrawal:changes in apoptosis-related genes. Biol Reprod,1999,60 (2):461-470.
[23]Hikim,A.P. Involvement of apoptosis in the induction of germ cell degeneration in adult rats after gonadotropin-releasing hormone antagonist treatment. Endocrinology,1995. 136(6):2770-2775.
[24]Hikim P P,Wang C,Leung A,et al. Involvement of apoptosis in the induction of germ cell degeneration in adult rats after gonadotropin releasing hormone antagonist treatment. Endocrinology,1995,136,(6)1:2770.
[25]Lei Z M, Mishra S, Ponnuru P, et al. Testicular phenotype in luteinizing hormone recep tor knockout animals and the effect of testosterone replacement therapy. Biol Rep rod 2004,71 (5):1605-1613.
[26]崔毓桂,陈子庆,沙家豪.激素对精子发生的调控.中华男科学,2004,10(6)：465-467.
[27]Sinha Hikim AP,Lue Y,Diaz-Romero M,et al. DeciPhering the palthways of germ cell apoptosis [J].J Steroid Biochem Mol Biol,2003,85(2-5):175-182.
[28]Bakalska,M. Induction of male germ cell apoptosis by testosterone withdrawal after ethane dimethanesulfonate treatment in adult rats. Endocr Regul,2004.38 (3):103-110.
[29]Tesarik J,M.endoza C,Greco E. The effect of FSH on male germ cell survival and differentiation in vitro is mimicked by pentoxifylline but not insulin. Mol Hum Reprod, 2000,6(10):877-881.
[30]Nandi S,Banerjee PP,Zirkin BR. Germ cell apoptosis in the testes of Sprague-Dawley rats following testosterone withdrawal by ethane1,2-dimethane-sulfonate administ ration relationship to Fas. Biol Reprod,1999,61 (1):70-75.
[31]Mishra,D.P.and C.Shaha,Estrogen-induced spermatogenic cell apoptosis occurs via the mitochondrial pathway:role of superoxide and nitric oxide. J Biol Chem,2005.280 (7): 6181-6196.
[32]Yazawa,H.,I. Sasagawa,T.Nakada,Apoptosis of testicular germ cells induced by exogen-ous glucocorticoid in rats.Hum Reprod,2000.15(9):1917-1920.
[33]Koji T. Male germ cell death in mouse testes:possible involvement of Fas and Fas ligand. Med Electron Microsc,2001,34(4):213-22.
[34]Bhardwaj A&Aggarwal BB. Receptor-mediated choreography of life and death. J Clin Immunol,2003,23(5):317-32.
[35]Philchenkov AA. Caspases as regulators of apoptosis and other cell functions. Biochemi-stry (Mosc),2003,68(4):365-376.
[36]李宏军,俞莉章,郭应禄,等.Fas在正常睾丸组织及精原细胞瘤中的表达.中华男科学,2001,7(3)：150-153.
[37]Mizutani Y, Yoshida O, Bonavida B. Prognostic significance of soluble Fas in the serum of patients with bladder cancer. J Urol,1998,160 (2):571-576.
[38]Eguchi J,Koji T,Nomata K,et al. Fas-Fas ligand system as a possible mediator of sperma-togenic cell apoptosis in human maturation-arreseted testes.Hum Cell,2002,15(1): 61-68.
[39]Lee J, Richburg J H, Shipp EB, et al. The Fas system,a regulator of testicular germ cell apoptosis, is differentially up-regulated in Sertoli cell versus germ cell injury of the testis. Endocrinology,1999,140 (2):852-858.
[40]Richburg J H,Nanez A, Gao H. Participation of the Fas-signaling system in the initiation of germ cell apoptosis in young rat testes after exposure to mono-(2-ethylhe-xyl) phthalate. Toxicol Appl Pharmacol,1999,160 (3):271-278.
[41]Fujisawa M, Ishikawa T. Soluble forms of Fas and Fas ligand concentrations in the seminal plas ma of infertile men with varicocele. J Urol,2003,170(6Pt1):2363-2365.
[42]MacFarlane M,Williams C. Apoptosis and disease:a life or death decision.nat ure. EMBO reports,2004,5(7):674-678.
[43]Yamamoto CM,Sinha Hikim AP,Huynh PN,et al. Redistribution of Bax is an early step in an apoptotic pathway leading to germ cell death in rats,triggered by mild testicular hypothermia. Biol Reprod,2000,63(6):1683-1690.
[44]Oldereid NB,Angelis PD,Wiger R,et al. Expression of Bcl-2 family proteins and spontaneous apoptosis in normal human testis. Mol Hum Reprod,2001,7(5):403-8.
[45]Zhang ZH,Jin X.Zhang XS,et al. Bcl-2 and Bax are involved in experimental cryptorchidism-induced testicular germ cell apoptosis in rhesus monkey. Contraception, 2003,68(4):297-301.
[46]Eskes R,Desagher S,Antonsson B,et al. Bid induces the oligomerization and insertion of Bax into outer mitochondrial membrane. Mol Cell Biol,2000,20(3):929-935.
[47]Chandrasekaran,Y,J.H.Richburg. The p53 protein influences the sensitivity of testicular germ cells to mono-(2-ethylhexyl)phthalate-induced apoptosis by increasing the membrane levels of Fas and DR5 and decreasing the intracellular amount of c-FLIP.Biol Reprod,2005.72(1):206-13.
[48]Wunderlich M&Berberich SJ. Mdm2 inhibition of p53 induces E2F1 transactivation via p21.Oncogene,2002,21(28):4414-4421.
[49]Yin Y,DeWolf WC, Experimental cryptorchidism induces testicular germ cell apoptosis by p53-dependent and-independent pathways in mice. Morgentalor A,Biol Reprod,1998; 58:492-496
[50]Kilinc F,Guvel S,Kayaselcuk F,et al. p53 expression and apoptosis in varicocele in the rat tesis. J Urol,2004,172 (6 Ptl):2475-2478.
[51]Gao CF,Ren S,Zhang L,et al. Caspase-dependent cytosolic release of cytochrome c and membrane translocation of Bax in p53-induced apoptosis. Exp Cell Res,2001,265.(1): 145-151.
[52]Singh NP,Muller CH&Berger RE. Effects of age on DNA double-strand breaks and apoptosis in human sperm. Fertil Steril,2003,80(6):1420-1430.
[53]Erenpreisa J,Erenpreiss J,Freivalds T,et al. Toluidine blue test for sperm DNA integrity and elaboration of image cytometry algorithm. Cytometry,2003,52 (1):19-27.
[54]Jersmann HP,Ross KA,Vivers S,et al. Phagocytosis of apoptotic cells by human macroph-ages:analysis by multi-parameter flow cytometry. Cytometry,2003,51 (1):7-15.
[55]林本成,袭著革,张英鸽,等.应用流式细胞分析技术研究微纳尺度Si 0：对雄性大鼠睾丸生精细胞的毒性作用.生态毒理学报,2007.2(3)：322-326