| Di-(2-ethylhexyl)-phthalate(DEHP) is known as a typical environmental endocrine disruptor, with anti-androgen, estrogenic and anti-estrogenic effects. It can cause reproductive toxicity and neurotoxicity by disturbing the secretion of sex hormone, threatening the health of animals and humans seriously. The perinatal period and puberty are the critical period of the development of individual, are very sensitive to the outside environment hormone. Prepuberty is the transition between the period of childhood and puberty, at this stage, the gonads begin to develop and become mature gradually, it is very important for the development of the brain, the formation of behavior and the gender differentiation, to be known as the critical period of the development of emotional and cognitive behaviors. If DEHP exposure during this period, it is likely to interfere with the regulation of sex hormones on brain function and then influence the mood and behaviors.Social behavior is a series of actions between different individual animals communicate with each other in the same species, social behavior is relevant to the individual survival and reproduction. Social play behavior is the first step to launch social behavior, also is the key to social interaction. Social interaction is the ability of individual to society. The social novelty preference is a reflection of social cognition and social memory. The three social behaviors are the basic social skills which individuals should have. The change of the social behavior may be associated with the mood of the animals. Generally speaking, individuals in a buoyant mood have higher social behaviors, but there also have the opposite examples.The hippocampus is a brain region which is closely related to the cognitive function, the ability of learning and memory is fading along with the damage of hippocampus. At the end of the hippocampus is the amygdale, it is the key brain region to control emotional behavior, people with the damage of amygdale, show the mood disorders frequently, can’t express their feelings correctly, then, leading to the abnormal social behavior. The growth of the brain is sensitive to hormones. Sex hormones mainly play a role by sex hormone receptors. Estrogen receptor beta and androgen receptors are widely distributed in the hippocampus and the amygdale. The midbrain dopamine nervous system distributed in the amygdala is associated with the regulation of emotional behavior by amygdale. The dopamine transporter and dopamine receptors are important components of regulating dopamine neural systems, dopamine D2 receptor is associated with the development of emotion and behavior,In this paper, through the establishment of prepubertal DEHP exposed mice model, checking the influence of social behavior after prepubertal exposure to DEHP in mice by a variety of behavior models. And by examining the expression levels of androgen and estrogen receptors, the dopamine transporter, and the dopamine receptor 2 in the hippocampus and the amygdala to explore its action mechanism.Research contents:The influence of prepuberty DEHP exposure on the activity, anxiety, and social behavior of the puberty and adult male and female mice; the effects of prepuberty DEHP exposure on serum testosterone and estradiol levels in mice; the influence of prepubertal exposure to DEHP on the expression levels of dopamine transporter, dopamine D2 receptor, estrogen receptor (ER beta) and androgen receptor (AR).Research method:Prepubertal (28 to 42 days after birth, PND28-42) male and female mice exposure to DEHP (1,10,50,200 mg/kg/day) by gavage administration, at the same time set in the control group, respectively in PND42 and PND84, detected the activity of mice by the open field model, used elevated plus maze to test the anxiety of mice, used social play test to examine the social ability of mice and tested the ability of social interaction and social novelty preference by the three-chambered model. At the same time we took the hippocampus and the amygdala of PND42 and PND 84 mice (do not have a behavior test), examined the expression level of dopamine transporter, dopamine D2 receptor, estrogen receptor (ER beta) and androgen receptor (AR) by western blot, detected the serum testosterone and serum estradiol levels used radioimmunoassay.Research results1. Body Weight. Compared with the control group, after prepuberty DEHP exposure, in puberty:10 mg/kg/day DEHP significantly reduced the body weight of female mice (p<0.05),200 mg/kg/day DEHP increased the body weight of male mice significantly (p<0.05); in adulthood:1 mg/kg/day DEHP significantly increased the body weight of female mice (p< 0.01).2. Serum hormone levels and reproductive organs. After prepubertal exposure to DEHP, during puberty:50 and 200 mg/kg/day DEHP decreased the testicle coefficient of male mice significantly (p<0.05, p<0.01); in adulthood:200 mg/kg/day DEHP significantly shorten the anogenital distance of male mice (p<0.01).3. Behavior test results:1) Open field test. After DEHP exposure, compared with the control group, in puberty: male mice in 10-200 mg/kg/day significantly increased the run number of 1 min, and 5 min (p<0.05, p<0.01), increased the grooming number between 1 and 200 mg/kg/day (p<0.05) and the stand number at 200 mg/kg/day significantly (p<0.05); 50 and 200 mg/kg/day DEHP exposure increased the 5 min running number and stand number significantly (p<0.05, p<0.01), and significantly reduced the grooming number after DEHP exposure at 200 mg/kg/day (p<0.05) in female mice. The open field test of adulthood mice was still affected by DEHP exposure. In male mice:the 5 min run number was significantly increased at 50 and 200 mg/kg/day DEHP exposure (p<0.05), at 200 mg/kg/day DEHP exposure decreased the grooming number significantly (p<0.01), DEHP exposure at 10-200 mg/kg/day reduced the stand number (p<0.01); in female mice, after DEHP exposure at 10 and 50 mg/kg/day reduced the 5 min running number obviously (p<0.05, p<0.01), obviously increased the grooming number at 1 and 50 mg/kg/day DEHP exposure (p<0.05, p<0.01),10 mg/kg/day DEHP exposure reduced the stand number obviously (p<0.05).The results showed that DEHP exposure during prepuberty can increase the activity of the male mice, but age differences influence on female mice, characterized by increased their activity in puberty, inhibit its activity in adulthood.2) Elevated plus maze test. The behavior of mice in elevated plus maze test was influenced by prepuberty DEHP exposure. The performance of puberty mice:1 and 50 mg/kg/day DEHP exposure significantly increased the time in open arms (p<0.05, p<0.01), and DEHP exposure at 10-200 mg/kg/day increased the probe number in the central region significantly (p<0.01, p<0.05) in male mice; 1 or 10 mg/kg/day DEHP exposure significantly decreased the time and frequency into the open arms (p<0.05, p<0.01),50 and 200 mg/kg/day significantly increased the number of central probe (p<0.01) in female mice. In adulthood, after DEHP exposure,1～50 mg/kg/day the time in the open arms was increased significantly(p<0.05, p<0.01),200 mg/kg/day significantly reduced the number of central probe (p<0.01),1 mg/kg/day significantly reduced its total numbers (p<0.05) in male mice; in female mice,1-200 mg/kg/day DEHP exposure reduced the time and frequency into open arms significantly (p<0.05). The above results showed that the influence of DEHP exposure on anxiety levels of male and female mice is different, DEHP exposure can reduce the anxiety of male mice, but increase the level of anxiety in the female mice.3) Social play test. For the time spent in social play:in puberty, exposure to DEHP significantly reduced the time spent in social play of the females at 1 and 50 mg/kg/d (p<0.05) but extended that of the males when compared to the same sex controls (p<0.01). In adult, DEHP significantly reduced the time spent in social play of both females at 200 mg/kg/d (p<0.05) and males at 1 and 200 mg/kg/d (p<0.01).For the time spent in social investigation:In puberty, DEHP at 1-200 mg/kg/d significantly reduced the time spent in social investigation in the females (p<0.05 and p<0.01) but extended that at 50 mg/kg/d in the males respectively when compared to the same sex controls (p<0.01). In adult, DEHP at 1,50, and 200 mg/kg/d significantly reduced the time spent in social investigation in males, but no significant effect was found in females. These results suggest that early pubertal exposure to DEHP inhibited social play and social investigation of pubertal females, but this effect on social investigation was disappeared after adult; while in males, DEHP enhanced social play and social investigation during puberty but inhibited these behaviors in adult.4) Sociability test. In pubertal females, DEHP exposure, especially at 200 mg/kg/d, significantly reduced the time spent in sniffing the stranger mouse and increased the time spent in the left chamber containing empty cylinder and extended the time spent in sniffing the empty cylinder (at 1 mg/kg/d, p<0.05) when compared to the same sex vehicle controls. In pubertal males, DEHP, especially at 10 and 50 mg/kg/d, significantly extended the time spent in sniffing the stranger (p<0.05) and in right chamber containing the stranger mouse (p<0.05) but significantly reduced the time spent in sniffing empty cylinder (at 10 mg/kg/day, p<0.05), in left chamber when compared to the same sex controls. In adult, DEHP at 1 mg/kg/d significantly reduced the time spent in the right chamber (p<0.05) but increased the time spent in the left chamber (p<0.05) of females when compared to the same sex controls, but has no influence in adult males. The results showed that the early pubertal DEHP exposure can increase the sociability in pubertal male mice, but in the high-dosage group (200 mg/kg/day) can reduce the sociability in pubertal females. It has little impact on adult mice, just 1 mg/kg/day DEHP exposure can inhibit the sociability of females.5) Social novelty preference task. In pubertal females, DEHP at 1～50 mg/kg/d significantly decreased the time spent in the right chamber containing familiar mouse (p<0.01) and in sniffing familiar mouse (p<0.05) when compared to the corresponding vehicle controls. In pubertal males, on the contrary, DEHP at 200 mg/kg/d significant reduced the time spent in sniffing unfamiliar mouse (p<0.05) when compared to the same sex controls. In adult, the effect of DEHP on novelty preference also exhibited sex-specific characteristics. DEHP at 1 or 50 mg/kg/d significantly increased the time spent in the left chamber (p<0.01) or in sniffing unfamiliar mouse (p<0.01) of females, but DEHP at 1 mg/kg/d decreased the time spent in the left chamber (p<0.01) and in sniffing unfamiliar mouse (p<0.05) of males, when compared to the same sex controls. These results indicated that early pubertal DEHP exposure enhanced the social novelty preference of females but inhibited that of males (females< males).4 Further Western blot analysis showed, after prepuberty DEHP exposure, different doses significantly lowered the expression of ER beta of male and female mice in hippocampus and amygdala in different degrees; the expression of AR significantly increased in hippocampus and amygdala of puberty male mice. DEHP exposure had no significant influence on dopamine transporter (DAT) but downgraded the expression of dopamine receptor D2 in hippocampus and amygdala.Conclusion:Prepubertal DEHP exposure can affect the anxiety and social behavior of male and female mice in puberty and adulthood and has dose, gender and age differences. Different behaviors have different levels of sensitivity to DEHP exposure, and the change of mood may be related to the change of social behavior. When DEHP change the social behavior, the expression of the estrogen and androgen receptors and the dopamine receptor changed. So we speculate that the influence on social behavior of DEHP may be related to the disturbance of sex hormone receptors, at the same time, the dopamine nervous system may also be involved in the regulation of social behavior. |
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