The Combined-effect Of Dioctyl Phthalate And Chloroform On Reproductive Toxicity In Male Mouse

ObjectivesDOP is widely used as a plasticizer and solvents in plastics and decorative materials, and the usage is increasing year by year, due to easily being dissolved, released and having environmental estrogen-like effect, so its pollution situation and damage to the human are getting more and more attention. Chloroform as the main component of chlorination by-products ,its toxicity has been extensively studied, but at present the report about the combined effect of DOP and chloroform.This study was designed to observe the reproductive toxicity on male mice when they are exposed to DOP and chloroform by measuring the enzyme activity of testicular germ cells, lipid oxidation, testosterone and other hormones'level ,and initially discuss the mechanism of the two substance injuring the body.MethodsThe experiment was grouped according to the analytical method of combined effect of -2×2 factorial experimental design.There were two factors, DOP and chloroform,and each factor had two levels: exposure and unexposure.So there are four combinations in all, including :①exposed by neither DOP nor chloroform;②exposured to DOP only;③e xposured to chloroform only;④exposed by both DOP and chloroform. According to the principle of randomization, the mice were divided into control group, DOP exposure group, combined exposure group .Control group, DOP exposure group, chloroform exposure group, and combined exposure groups were exposed adopting administration by gavage, the administration dose were: DOP group 540mg/Kg, chloroform group 112mg/Kg, the combine group DOP270mg/Kg + chloroform 56mg/Kg, adopting corn oil as excipients.Running administration 15 days,and then mice were killed, measured testosterone level in testis, germ cell enzyme activity and lipid peroxidation, detected the change of germ cell cycle, apoptosis percentage and the level of DNA damage.The datas were ananlyzed with SPSS 17.0 and results were demonstrated as x±s.and the 2×2 factorial designs was used to estimate combined effects of two toxic substances.Results1,The combined effect of DOP and chloroform on the weight and organ indices of testis.15 days after exposure, the weight of chloroform group was significantly less than the control group (P <0.05), the testis indices of DOP group is less than the control group, the difference was significant (P <0.05).2,The combined effect of DOP and chloroform on the testis cell enzyme activity.The result of G-6-PD showed: the activity of chloroform group was lower than the control group (P <0.05), while the DOP group and the combined group's activity were higher than control group(P<0.05).The LDH activity of chloroform and DOP group were significantly decreased than control group(P<0.05); combined exposuring of DOP and chloroform could take interaction.3,The combined effect of DOP and chloroform on the testis lipid peroxidation.The MDA content in testis tissue: DOP group was lower than control group (P <0.05), while the chloroform group was higher than control group (P<0.05), there was no significant difference between the combined group and the control group (P> 0.05); the SOD activity of DOP group was significantly lower than control group (P <0.05), while the chloroform group and control groups had no significant difference (P>0.05); the GSH-Px activity of DOP groups was lower than the control group (P<0.05), while the chloroform group was higher than the control group(P<0.05), combined exposuring of DOP and chloroform could take interaction on GSH-Px activity.4,The combined effect of DOP and chloroform on the testosterone level of testis tissue.The testosterone level of DOP group was significantly lower than the control group,while the chloroform group was higher than the control group(P <0.05), combined exposuring of DOP and chloroform could take interaction5,The combined effect of DOP and chloroform on the level of LH and FSH in serum.The LH and FSH level of DOP and chloroform group were significantly lower than the control group (P <0.05), the combined group decreased more markedly, suggesting that the two could take interaction, but we couldn't confirm it's the role of synergistic or additive. 6,The combined effect of DOP and chloroform on the testis cell apoptosis and cell cycle.The percentage of G0/G1 phase cells in all experimental groups was significantly decreased than the control group(P<0.05), and there was interaction between the two substances; the S phase cell percentage was significantly higher than the control group (P <0.05). the apoptosis percentage of DOP and chloroform group were both higher than the control group(P<0.05).7,The combined effect of DOP and chloroform on the DNA damage.0 grade DNA Damage: the DOP and chloroform group were both significantly lowerⅠa ndⅡgrade DNA Damage: the DOP and chloroform group were both significantly higher than the control group(P <0.05); and the combined exposure could no significant interactions.8,Histopathological examination on testis of mice.In the DOP group, the lesion of seminiferous tubules was spotty, some seminiferous epithelial cells arranged loosely, the spermatogenic cells on the tubule wall significantly reduced, and the mature sperm in the also reduced, focal Leydig cell hyperplasia and interstitial edema, lumen of individual seminiferous tubules could find multinucleated giant cells. The chloroform group had the similar lesions with the DOP group, but the degree was lower than DOP group. The combined group manifest more serious lesions than the DOP and chloroform group.ConclusionsThe results show that the combined effect of DOP and the chloroform could cause the LDH activity, the LH and FSH level in the serum decrease, the apoptosis rate of testicular cells and the percentage of DNA damage increased, DNA synthesis in testicular cells decreased and S phase was blocked, there is a significant extent interactions between the two substances.