Effect Of Lactational Exposure To Low Level 2,3,7,8-tetrachlordibenzo-p-dioxin On The Neurodevelopment Of Mice Offspring

ObjectiveThe contaminant 2, 3, 7, 8-tetrachlordibenzo-p-dioxin (TCDD) is one of the most toxical compounds and ranked asⅠlevel carcinogen by International Agency for Research on Cancer (IARC). It is considered by many agencies such as WHO that TCDD belongs to the category of high carcinogenicity, but the risk of non-carcinogenicity toxic effect on human health is higher than that of carcinogenicity toxic effect in present exposure background. In addition, structural stability and long half-life period lead to TCDD staying or accumulating in human body after once or long-time exposure. As a result, TCDD induce severe toxic effects to the organism.For mice offspring are hypersensitive to TCDD, in this study, the female mice got TCDD by intraperitoneal injection immediately after parturition and the offspring got by breast feeding. The present study would identify the toxic effect of lactational exposure to low level TCDD on mice offspring by observing body weight, neural development, learning and memory ability, the histological changes and expression of cytochrome P450 1A1 enzyme (CYP1A1), apoptosis gene B-Cell lymphoma/leukemia 2 (Bcl-2) and Bcl-associated x protein (Bax) in hippocampus and cerebral cortex tissue. The results would provide evidence for the study about the chronic nervous system effect and mechanism of lactational exposure to low level TCDD on mice offspring.Methods1 Animals model and groups4-month-old mature Kunming mice with 24 female and 8 male were grouped by 3:1. After parturition, the female mice with 7~10 pups were retained, and 8 pups were adjusted as a unit, including 4 female and 4 male offspring. The others were eliminated. Then the female mice and its offspring, regarded as a unit, were divided into 5 groups randomly. In this study, there were 2 doses of treatment: 40μg and 20μg TCDD/kg body weight. Corresponding to 2 doses of TCDD treatment, there were 2 vehicle controls: 40μg and 20μg vehicle/kg body weight, and 1 animal control without any treatment. There were 5 groups totally and each group had 3 units (They noted as 40μg TCDD, 20μg TCDD, 40μg vehicle control, 20μg vehicle control and animal control group respectively). After parturition, the female mice were weighted and intraperitoneally injected. The female mice stop lactation on offspring postnatal days (PND) 21, and then offspring were fed on animal feeds. Mice offspring were killed on PND 35.TCDD was dissolved in methylbenzene with a concentration of 10μg/ml and a purity of 99% when purchased, and diluted with peanutoil by 1:4. The control vehicle was the mixed liquor with methylbenzene and peanutoil by 1:4.2 Measurement of mice offspring's body weight.3 Measurement of mice offspring's neurodevelopment indexes (surface fighting, negative geotaxis, forelimb grip force, air fighting, hindlimb splaying).4 Measurement of mice offspring's learning and memory ability (Y-maze test).5 Localization and semi-quantitative analysis of CYP1A1 protein expression in hippocampus and cerebral cortex tissue of mice offspring by immunohistochemistry.6 Detection of apoptosis in hippocampus and cerebral cortex tissue of mice offspring.6.1 Observation of structure changes in hippocampus and cerebral cortex tissue of mice offspring by H.E staining method.6.2 Localization and semi-quantitative analysis of Bcl-2 and Bax protein expression in hippocampus and cerebral cortex tissue of mice offspring by immunohistochemistry.6.3 Quantitative analysis of Bcl-2 and Bax protein expression in hippocampus and cerebral cortex tissue of mice offspring by flow cytometry.Results1 The effect of TCDD on the body weight of mice offspring There was no difference in the average body weight of mice offspring between 5 groups when the offspring were born (P>0.05). On PND 7, the average body weight in 20μg TCDD group decreased compared with 20μg vehicle control and animal control groups (P<0.01). Along with the time of breast feeding, the average body weight in 40μg and 20μg TCDD groups decreased compared respectively with vehicle control and animal control groups (P<0.01), and that in 40μg TCDD group also decreased compared with 20μg TCDD group (P<0.05). After mice offspring were weaned (on PND 28 and PND 35), the average body weight in two TCDD groups also decreased compared with vehicle control and animal control groups (P<0.01).2 The effect of TCDD on the neurodevelopment of mice offspringThe time of surface fighting and negative geotaxis in two TCDD groups were longer than those in vehicle control and animal control groups (P<0.01). The time of forelimb grip force and the distance of hindlimb splaying in two TCDD groups were shorter than those in vehicle control and animal control groups (P<0.01). The correct rates of air fighting in two TCDD groups also decreased compared respectively with vehicle control and animal control groups (P<0.01).3 The effect of TCDD on learning and memory ability of mice offspringIn Y-maze test, the abilities of learning (A), memory (B) and the memory retaining rate (C) were decreased in two TCDD groups compared respectively with vehicle control and animal control groups (P<0.01). 4 The effect of TCDD on CYP1A1 protein expression in hippocampus and cerebral cortex tissue of mice offspringCYP1A1 protein expressed mainly in the cytochylema of pyramidal cell in hippocampus and cerebral cortex tissue of mice offspring. The average optical density of CYP1A1 protein increased in two TCDD groups compared respectively with vehicle control and animal control groups (P<0.01). The female offspring's CYP1A1 protein expression was higher than the male offspring's, but there was no statistical significance of the difference (P>0.05).5 The effect of TCDD on apoptosis in hippocampus and cerebral cortex tissue of mice offspringLactational exposure to low level TCDD led to pyramidal cell apoptosis such as disordered structure, cell body crenation and chromatin agglutination in hippocampus and cerebral cortex tissue of mice offspring.Bcl-2 protein expressed mainly in the cytochylema of pyramidal cell in hippocampus and cerebral cortex tissue of mice offspring. It had statistical difference between the female and male offspring in vehicle control and animal control groups, and the average optical density of Bcl-2 protein of female offspring was higher than that of male offspring (P<0.01). Lactational exposure to low level TCDD made Bcl-2 protein expression decreased both in female and male offspring(P<0.01), but the female offspring's Bcl-2 protein expression was still higher than the male offspring's (P<0.01). Bax protein expressed mainly in pyramidal cell membrane and cytochylema in hippocampus and cerebral cortex tissue of mice offspring. It had no statistical difference between the female and male offspring in vehicle control and animal control groups (P>0.05). Lactational exposure to low level TCDD made Bax protein expression increased both in female and male offspring(P<0.01), and the female offspring's Bax protein expression was higher than the male offspring's (P<0.05).The ratio of Bcl-2/Bax decreased significantly in two TCDD groups compared respectively with vehicle control and animal control groups (P<0.01). It suggested that TCDD would induce pyramidal cell apoptosis in hippocampus and cerebral cortex tissue of mice offspring. Furthermore, the Bcl-2/Bax ratio of male offspring was lower than that of female offspring (P<0.01). It suggested that the apoptosis rate of male offspring was higher than that of female.Conclusion1 Lactational exposure to low level TCDD would delay the body weight gain of mice offspring. This adverse effect was persistent and did not vanish when stopping TCDD intake.2 Lactational exposure to low level TCDD would step down the function of nervous reflex, decrease the ability of movement and coordination, degrade the tension of muscle and delay the development of nervous system of mice offspring. And the more dose TCDD intook, the more obvious the adverse effect was.3 Lactational exposure to low level TCDD would impair the spatial learning and memory abilities and damage the danger-avoiding capability of mice offspring.4 Lactational exposure to low level TCDD would increase CPY1A1 protein expression in hippocampus and cerebral cortex tissue of mice offspring. It demonstrated that blood-brain barrier had a limited effect on preventing TCDD from entering the brain tissue, so TCDD could damage hippocampus and cerebral cortex tissue. It might be one of the important mechanisms of TCDD delaying neural development and impairing learning and memory ability of mice offspring.5 Lactational exposure to low level TCDD would induce pyramidal cell apoptosis, decrease Bcl-2 protein expression, increase Bax protein expression, decrease the Bcl-2/Bax ratio in hippocampus and cerebral cortex tissue of mice offspring. The ratio changes of Bcl-2 apoptosis gene family might be one of the important mechanisms of TCDD inducing abnormal apoptosis, and might also be one of the mechanisms for TCDD delaying neural development and impairing learning and memory ability. In addition, the sexual difference in the Bcl-2/Bax ratio in hippocampus and cerebral cortex tissue might be one of the causes why there were significant discrepancies in TCDD sensitivity and impairment between the female and male offspring.