Effect Of Lactational Exposure To Low Level Of TCDD On The Expression Of CYP1A1 And Apoptosis In The Heart, Kidney And Testes In 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). TCDD can accumulate in human body after long-time exposure. As a result, TCDD induce severe toxic effects on the organism. By far now, more attention is devoted to research the mechanism of acute toxicity and the effect of pregnant exposure TCDD on offspring. However, the effect of lactational exposure to low level of TCDD is ignored.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, 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 heart, kidney and testes tissue. The results would provide evidence for the toxic effects of lactational exposure to low level of TCDD on mice offspring. Methods1 Animals4-month-old mature Kunming mice with 24 female and 8 male were grouped by 3:1. After parturition, the female mice with 8-10 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 and the final concentration is 2μg. 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. 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 35.4 Measurement of mice offspring's development indexes including body weight, the weight of kidney and testes, descending time of testes.5 CYP1A1 protein expression in heart, kidney and testes tissue of mice offspring.6 Detection of apoptosis in heart, kidney and testes tissue of mice offspring6.1 Observation of structure changes in heart, kidney and testes tissue of mice offspring by H.E staining method.6.2 Localization and semi-quantitative analysis of Bcl-2 and Bax protein expression in heart, kidney and testes tissue of mice offspring by immunohistochemistry.Results1 The effect of TCDD on the body weight of mice offspringThere 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<0.05). After 7 days, the average body weight in 40μg and 20μg TCDD groups decreased compared respectively with vehicle control and animal control groups (P<0.05), 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<0.05).2 The effect of TCDD on the kidney's weight of mice offspringThe absolute weight of kidney in two TCDD groups of the female was decreased significantly compared with those in vehicle control and animal control groups (P<0.05). But only the absolute weight of kidney in 40μg TCDD group of the male were decreased significantly compared with vertical control group (P<0.05). The relative weight of kidney in two TCDD groups of the female and male were no significant difference with the vehicle control (P>0.05). However, the relative weight of kidney in two TCDD groups of the female were also decreased significantly compared respectively with animal control groups (P<0.05). The absolute weight of kidney and relative weight of kidney in 40μg TCDD groups of the female and male were no significant difference with the 20μg TCDD groups (P>0.05). All the indexes of female were lower significantly than the male's (P<0.05).3 The effect of TCDD on the testes weight of mice offspringThe absolute weight and relative weight of testes in two TCDD groups was decreased compared respectively with vehicle control and animal control groups. Meanwhile, 40μg TCDD groups was lower than those in 20μg TCDD groups, but there was no significant difference (P>0.05). Only the 40μg TCDD groups were decreased significantly compared with the vehicle control groups (P<0.05). 4 The effect of TCDD on descending time of testes of offspring miceThe descending time of testes in two TCDD groups was latter significantly than those in vehicle control and animal control groups and had significant difference compared respectively with vehicle control and animal control groups (P<0.05). The descending time of testes in 40μg TCDD groups was latter significantly than those in 20μg TCDD groups (P<0.05).5 The effect of TCDD on CYP1A1 protein expression in heart, kidney and testes tissue of mice offspringCYP1A1 protein can express mainly in the vascular endothelial cell, kidney and testes of mice offspring. Lactational exposure to low level of TCDD would induce CYP1A1 protein in myocardium increased in TCDD groups (P<0.05), but there was no significant difference between male and female (P>0.05). Lactational exposure to low level of TCDD would induce CYP1A1 protein in kidney increased in TCDD groups (P<0.05), and the female offspring's CYP1A1 protein expression was higher significantly than the male offspring's (P<0.05). Meanwhile lactational exposure to low level of TCDD also induced CYP1A1 protein expression in testes increased significantly in TCDD groups (P<0.05).6 The effect of TCDD on morphologic and Bcl-2 or Bax protein expression in heart, kidney and testes tissue of mice offspring Lactational exposure to low level of TCDD led to vascular endothelial cell apoptosis such as disordered structure, cell body crenation in heart of mice offspring. Lactational exposure to low level of TCDD led to the number of glomerular decreased and the dimension of glomerular increased. Meanwhile, inflammatory cells were also found in kidney. Meanwhile, cell body crenation of testes was also increased.Bcl-2 protein mainly expressed in vascular endothelial cell in heart of mice offspring, which can inhibit the cell apoptosis. Lactational exposure to low level of TCDD led to the Bcl-2 protein expression of 40μg TCDD groups in heart of mice offspring were no significant difference with the 20μg TCDD groups (P>0.05). Bcl-2 protein mainly expressed in glomerular of kidney. Lactational exposure to low level of TCDD led to the Bcl-2 protein expression and the Bcl-2 protein expression in 40μg TCDD groups in kidney of mice offspring were lower significantly than those in 20μg TCDD group's (P<0.05). Bcl-2 protein also expressed in testes and Lactational exposure to low level of TCDD led to the Bcl-2 protein expression of 40μg TCDD groups in testes of mice offspring were no significant difference with the 20μg TCDD groups (P>0.05). Bcl-2 protein expression in two TCDD groups were lower significantly than those in vehicle control and animal control groups (P<0.05). Bcl-2 protein expression in heart and kidney were no significant difference between male and female (P>0.05).Bax protein expressed mainly in cardiac muscle cell of mice offspring. Lactational exposure to low level of TCDD led to the Bax protein expression of 40μg TCDD groups in heart of mice offspring were no significant difference with the 20μg TCDD groups (P>0.05). Meanwhile, there was no significant difference between male and female (P>0.05). Bax protein can express in glhemerular and renal tubules of the kidney. Lactational exposure to low level of TCDD, the Bax protein expression in 40μg TCDD groups in kidney of mice offspring were higher significantly than those in 20μg TCDD group's (P<0.05) and the female offspring's Bax protein expression was higher significantly than those of the male offspring (P<0.05). Bax protein also expressed in testes and Bax protein expression in 40μg TCDD groups were higher significantly than those in 20μg TCDD groups (P<0.05).Bax protein expression in two TCDD groups were higher significantly than those in vehicle control and animal control groups (P<0.05).In heart, kidney and testes, the ratio of Bcl-2/Bax decreased significantly in two TCDD groups of mice offspring compared respectively with vehicle control and animal control groups (P<0.05), there was no significant difference between male and female (P>0.05). Lactational exposure to low level of TCDD led to the the ratio of Bcl-2/Bax of 40μg TCDD groups in kidney of mice offspring were higher significantly than 20μg TCDD group's (P<0.05).ConclusionLactational exposure to low level of TCDD delaied the body weight gain of mice offspring;inhibited the weight of kidney. The toxic effects are responsible for the growth retardation of kidney;hampered the weight of testes. TCDD can display the toxic effect and damage the development of testes;decreased the descending time of testes,disturb the endocrine system and affect the function of testes;increased CYP1A1 protein expression in heart, kidney and testes. TCDD has the relation to the CYP1A1 protein expression;induced the cell apoptosis in heart, kidney and testes. Meanwhile, Lactational exposure to low level of TCDD led to decrease of Bcl-2 protein expression and the increase of Bax protein expression. The change of of Bcl-2 Protein expression is correlated with accelerated apoptosis. In addition, the ratio of Bcl-2/Bax was also decreased and the ratio of Bcl-2/Bax in female offspring was higher than male offspring.