| Background:Recently, there has been public concern over the potential adverse effects of environmental factors on the male reproductive system. Phthalate esters (PAEs), which are one type of major endocrine disruptors, are commonly used as plasticizers for polyvinyl chloride plastics. The PAEs can interfere with the hypothalamus-pituitary-testicular axis balance and affect the process of spermatogenesis directly or (and) indirectly. Puberty is the key phase for physical development. If adolescent testis suffered some infaust environmental factors during this phase, their reproductive function and even individual growth and development would be disturbed. Recent reports have emphasized the need for testing combinations (two or more) of phthalates to better assess the health risks of these chemicals. Di-(2-ethylhexyl) phthalate (DEHP) and di(n-butyl) phthalate(DBP) are most commonly used and ubiquitous compounds in the world, and the major route of exposure in the general population is the ingestion of food and water contaminated with PAEs. So we aim to investigate the effects of co-administered DEHP and DBP on the reproductive endocrine system in pubertal rat, which are useful to prevent, delay or abate the damages by above factors.Objective:1) To explore the changes of mating behavior in male rats exposure to PAEs. 2) To investigate the histological changes of testis and spermatogenesis, and analyze the reproductive hormones changes after PAEs exposure. 3) To examine the spermatogenesis related enzymes activity changes and the oxidative stress and anti-oxidative system disorders after PAEs exposure. 4) To investigate the changes of testicular copper, zinc content and copper/zinc values by administering the PAEs compounds.Materials and methods:Pubertal male Sprague-Dawley rats (6 weeks old) were housed under specific conditions on a constant 12-hour light/dark cycle and at a controlled temperature of 22±2℃. Standard pellet food and tap water were available ad libitum. All male rats were randomly divided into 4 groups with 14 in each. The combination of DEHP and DBP (dissolved in corn oil) was gavaged at the dosage of 0 (control), 375, 750, 1500 mg/(kg.d). After 2 weeks exposure, 7 rats in per group were killed by decapitation. On the day of 22~24 after exposure, the male rats mating behaviors were observed and recorded. After 4 weeks exposure, another 7 rats in per group were killed. The individuals were weighted everyday and the dosage adjusted accordingly. After decapitation, the samples were collected, and the seminiferous tubular diameter and height of germinal epithelium were examined. The changes of spermatogenic cell were analyzed by flow cytometry method. The contents of serum testosterone (T) and estradiol (E2) were examined by radioimmunity (RIA). The serum and testicular homogenate ACP, SDH, SOD and MDA activities were assayed by ultraviolet spectrophotometer, and the homogenate copper, zinc contents were detected by atomic absorption spectrophotometry.Results:1. The sexual behavior changes: Ejaculation was seen in 6 rats in control and 750 mg/kg.d group, 7 rats in 375 mg/kg.d group and 5 rats in 1500 mg/kg.d group within 30min. Animals exposed to the dosage of 1500 mg/kg.d displayed an increased intromission latency and need a larger number of intromissions to achieve ejaculation when compared with controls (P <0.05).2. Histological changes of testicular: After 2 weeks exposure, seminiferous tubular diameter was decreased at the dose of 750 mg/kg.d (P <0.05), and significantly decreased (P <0.01) at the dose of 1500 mg/kg.d compared with the controls. The seminiferous tubular diameter was reduced at all dosages when exposured 4 weeks. The height of germinal epithelium was decreased in all experiment groups compared with controls.3. Spermatogenic cell: The percentage of hypoploid cells was increased when gavaged at the dosage of 750 mg/kg.d and 1500 mg/kg.d after 2 and 4 weeks exposure. The percentages of 1C cells and 4C cell were decreased and the percentage of 2C cell was increased as the dosage increased. The cell cycle analyzed that the proportion of G0/G1 phase was increased, although the G2/M phase cells were decreased as the dosage increased when exposed 2 and 4 weeks.4. The changes of T, E2 and NO content in serum: The total serum T levels were decreased at the dosage of 750 mg/kg.d and 1500 mg/kg.d when gavaged 2 weeks consecutively, although the serum E2 levels were increased at the same dosage when exposed 4 weeks DEHP and DBP mixture. The NO levels were increased at the dosage of 1500 mg/kg.d when compared with the controls (P <0.05).5. The serum and testicular homogenate ACP, SDH, SOD and MDA activities: After 2 weeks exposure, the serum SOD activity was decreased at the dosage of 1500 mg/kg.d (P <0.05); The testicular homogenate ACP, SOD, MDA activities were different from that in controls (P <0.05). After 4 weeks exposure, the serum ACP activity was decreased at the dosage of 750 mg/kg.d and 1500 mg/kg.d when compared with the controls. Significant difference was seen in SDH activity when administered 750 mg/kg.d phthalate esters compounds. Also, testicular homogenate ACP, SDH, SOD and MDA activities in experimental groups were significant different from that in controls.6. Testicular copper, zinc contents and copper/zinc value changes: After 2 weeks exposure, the zinc content in testis and the copper/zinc value were different from that in controls at the dosage of 1500 mg/kg.d group (P <0.05). After 4 weeks exposure, the testicular zinc content was decreased and the copper/zinc value was increased when the dosage was 1500 mg/kg.d.Conclusions:1. Phthalate esters can affect male rats sexual behavior, which may related to the disorders of the reproductive hormones and NO levels.2. Phthalates can seriously affect the growth and development of pubertal testis. Also, the integrity of seminiferous tubular structure can be damaged, which may result in spermatogenic dysfunction.3. The changes of spermatogenesis related enzyme activity may play key important roles in phthalate esters induced spermatogenic dysfunction mechanisms. The oxidative stress and anti-oxidative system disorders may be important for the testicular damages by phthalates.4. Testicular changes of copper, zinc contents and copper/zinc values may be significant for the damages induced by phthalates.
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