Effect Of Lactational Exposure To Low Level Of 2, 3, 7, 8-tetrachlordibenzo-p-dioxin On The Expression Of CYP1A1 And Apoptosis In The Immune Organ Of Mice Offspring

ObjectiveDioxinlike chemicals include polychlorinated dibenzo -p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and polychlorinated biphenyls (PCBs). The 2, 3, 7, 8 -tetrachlordibenzo-p-dioxin (TCDD) is the most toxical compound of them. TCDD, with stabile structure, was ingested into human body through food chain and accumulated in fat of animals and persons, forming serious threat to human health. Dioxin has been included in the list of monitoring objects in food part of the global environment monitoring program as a new environmental pollutant by World Health Organization.For the effect of lactational exposure to TCDD on offspring has been little, 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 of TCDD on mice offspring by observing body weight, thymus and spleen development, 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 thymua and spleen tissue. The results would provide evidence for the study about the immune organ effect and mechanism of lactational exposure to low level of TCDD on mice offspring.Methods1 Experimental animal4-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.2 ChemicalsTCDD 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 1:4.3 GroupsThe female mice and its offspring, regarded as a unit, were divided into 5 groups randomly (each group had 3 units): 40μg and 20μg TCDD/kg body weight,40μg and 20μg vehicle/kg body weight, and 1 animal control without any treatment. 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 35.4 Measurement of mice offspring's body weight.5 Measurement of mice offspring's immune organ development indexes (the weight and quotient of thymus and spleen).6 Localization and semi-quantitative analysis of CYP1A1 protein expression in thymus and spleen tissue of mice offspring by immunohistochemistry.7 Detection of apoptosis in thymus and spleen tissue of mice offspring.7.1 Observation of structure changes in thymus and spleen of mice offspring by H.E staining method.7.2 Localization and semi-quantitative analysis of Bcl-2 and Bax protein expression in thymus and spleen tissue of mice offspring by immunohistochemistry.Results1 The effect of TCDD on the body weight of mice offspring There was no difference in the 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<0.05). 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<0.05), and the weight in 40μg TCDD group also decreased compared with that in 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 that in vehicle control and animal control groups (P<0.05).2 The effect of TCDD on immune organs of mice offspring2.1 The effect of TCDD on the thymus tissue of mice offspringThe weight of the thymus of the male and female offspring in 40μg and 20μg TCDD groups was no significant difference compared with the corresponding vehicle control groups. The quotient of the male offspring in 40μg and 20μg TCDD groups was significantly increased compared with the corresponding vehicle control groups (P<0.05), but the quotient of female offspring was no significant difference compared with the corresponding vehicle control groups.2.2 The effect of TCDD on the spleen tissue of mice offspring The weight of the thymus and spleen in 40μg and 20μg TCDD groups was no significant difference compared with the corresponding vehicle control groups. The quotient of immune organs of male offspring in 40μg TCDD groups was singnificantly increased compared with 20μg TCDD and corresponding vehicle control groups(P<0.05).The above result suggested that lactational exposure to TCDD could increase the quotients of the male offspring's immune organs, but there was no significant difference in that of the female offspring's.3 The effect of TCDD on CYP1A1 protein expression in the thymus and spleen tissue of mice offspringCYP1A1 protein expressed mainly in the mitochondrion and endoplasmic reticulum of lymphocyte in thymus and spleen tissue of mice offspring. The average optical density of CYP1A1 protein increased in 40μg and 20μg TCDD groups compared respectively with vehicle control and animal control groups (P<0.05). The female offspring's CYP1A1 protein expression was higher than the male offspring's(P<0.05).4 The effect of TCDD on apoptosis in the thymus and spleen tissue of mice offspringLactational exposure to low level of TCDD led to lymphocyte apoptosis such as structural disorder, cell crenation and chromatin agglutination in 40μg and 20μg TCDD groups of mice offspring's thymus and spleen tissue.Bcl-2 protein was expressed mainly in the cytochylema of lymphocyte in the thymus and spleen tissue of mice offspring. Lactational exposure to low level of TCDD made Bcl-2 protein expression decreased both in mice offspring(P<0.05), but the female offspring's Bcl-2 protein expression was still higher than the male offspring's (P<0.05).Bax protein expressed mainly in lymphocyte membrane and cytochylema in the thymus and spleen tissue of mice offspring. Lactational exposure to low level of TCDD made Bax protein expression increased both in mice offspring(P<0.05), 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 40μg and 20μg TCDD groups compared respectively with vehicle control and animal control groups (P<0.05). It suggested that TCDD would induce lymphocyte apoptosis in thymus and spleen tissue of mice offspring.Conclusion1 Lactational exposure to low level of TCDD delaied 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 of TCDD significantly increased the quotients of thymus and spleen in the male offspring mice, but there was no significant change in the female offspring mice. It needs further research on whether the immunotoxicity of TCDD on offspring mice has sexual difference or not.3 Lactational exposure to low level of TCDD increased CPY1A1 protein expression in thymus and spleen tissue of mice offspring, and female offspring's CPY1A1 protein expression is higher than male offspring's. The effect of TCDD on immune organs of mice offspring is difference between the female and male offspring.4 Lactational exposure to low level of TCDD induced lymphocyte apoptosis, decrease Bcl-2 protein expression, increase Bax protein expression, decrease the Bcl-2/Bax ratio in thymus and spleen 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 inhibiting immune function. In addition, the sexual difference in the Bcl-2 and Bax might be one of the causes why there were significant discrepancies in TCDD sensitivity and impairment between the female and male offspring.