Effct Of Trichloroisocyanuric Acid On The Structure And Function Of Testis In SD Rats

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 results1. Effect of TCCA on general conditions of RatsA 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 ratsIn 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 testisIn 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 testisThe 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 concentrationIn 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 ratsIn 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 ratsWe 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 ratsAfter 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.Conclusions1. 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.