Immunotoxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and Diethylstilbestrol (DES) in the Fetal Mouse Thymus and Liver

Abstract

Diethylstilbestrol (DES) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) have been identified as immunotoxicants causing thymic atrophy, thymocyte hypocellularity, phenotypic changes detected by CD4 and CD8 surface antigens, and progenitor T-cell targeting in the fetal mouse. We hypothesized that gestational exposure to these two compounds may lead to comparable histologic and gene expression alterations in the fetal mouse thymus and liver. Treatment of pregnant C57Bl/6 mice with doses of 5 or 10 ug/kg TCDD or 48 ug/kg DES by oral gavage on gestation days (gd) 14 and 16 severely depressed day 18 thymic cellularity. Histologic evaluation of day 18 fetal thymuses showed disruption of normal cortico-medullary architecture after TCDD or DES. Decreased thymocyte density was noted primarily in cortical zones where pyknotic cells were increased by either TCDD or DES treatment. Using day 18 thymocyte suspensions and flow cytometry, 7-AAD showed decreases in viable thymocytes from TCDD- or DES-treated fetal mice, and concomitant increases in thymocytes in early apoptosis. When thymocytes were co-identified with CD4 and CD8 cell surface antigen expression, enhanced apoptosis occurred in CD4+CD8+ phenotype after TCDD treatment. After DES exposure, increased apoptosis occurred in CD4-CD8- and CD4-CD8+thymocytes. Both TCDD and DES increased liver to body weight ratios and decreased ratios of hematopoietic to hepatic cells present. Cytomegaly was seen in hepatocytes of TCDD and DES treated animals, and these cells had more variable features, such as increased cytoplasmic basophilia and more prominent nucleoli. Real time quantitative PCR demonstrated that DES decreased c-jun, Bcl-2, and PKCalpha mRNA expression. These results suggest a shift away from proliferative activity and may reflect alterations noted predominantly in the hematopoietic population. TCDD increased c-jun mRNA expression with modest decreases in PKCalpha, and marked decreases in p53 also noted. Decreases in p53 suggest a pro-proliferative status of hepatic cells, while decreases in PKCalpha may indicate decreases in phosphorylation of substrates required for normal cell cycle progression. The increased c-jun suggests that this gene may play a role in the hepatocyte hyperplasia, as well as the diminution of hematopoiesis.