TCDD-induced Thymic Atrophy Research Articles

Targeted deletion of the aryl hydrocarbon receptor in dendritic cells prevents thymic atrophy in response to dioxin.

In nearly every species examined, administration of the persistent environmental pollutant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin, TCDD) causes profound immune suppression and thymic atrophy in an aryl hydrocarbon receptor (AhR) dependent manner. Moreover, TCDD alters the development and differentiation of thymocytes, resulting in decreases in the relative proportion and absolute number of double positive (DP, CD4+CD8+) thymocytes, as well as a relative enrichment in the relative proportion and absolute number of double negative (DN, CD4-CD8-) and single-positive (SP) CD4+CD8- and CD4-CD8+ thymocytes. Previous studies suggested that the target for TCDD-induced thymic atrophy resides within the hemopoietic compartment and implicated apoptosis, proliferation arrest of thymic progenitors, and emigration of DN thymocytes to the periphery as potential contributors to TCDD-induced thymic atrophy. However, the precise cellular and molecular mechanisms involved remain largely unknown. Our results show that administration of 10 µg/kg TCDD and 8mg/kg 2-(1H-indol-3-ylcarbonyl)-4-thiazolecarboxylic acid methyl ester (ITE) induced AhR-dependent thymic atrophy in mice on day 7, whereas 100mg/kg indole 3-carbinol (I3C) did not. Though our studies demonstrate that TCDD triggers a twofold increase in the frequency of apoptotic thymocytes, TCDD-induced thymic atrophy is not dependent on Fas-FasL interactions, and thus, enhanced apoptosis is unlikely to be a major mechanistic contributor. Finally, our results show that activation of the AhR in CD11c+ dendritic cells is directly responsible for TCDD-induced alterations in the development and differentiation of thymocytes, which results in thymic atrophy. Collectively, these results suggest that CD11c+ dendritic cells play a critical role in mediating TCDD-induced thymic atrophy and disruption of T lymphocyte development and differentiation in the thymus.

Resveratrol (3,5,4′‐trihydroxystilbene) protects pregnant mother and fetus from the immunotoxic effects of 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin

The "fetal basis of adult disease" hypothesis proposes that prenatal exposure to environmental stress can lead to increased susceptibility to clinical disorders later in life. In utero exposure of fetus to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) leads to alterations in T-cell differentiation in the thymus and increased susceptibility to autoimmune disease later in life. TCDD triggers toxicity through activation of aryl hydrocarbon receptor and severely affects maternal and fetal immune system during pregnancy. In this study, using a mouse model, we investigated if administration of resveratrol (RES; 3,5,4'-trihydroxystilbene) would inhibit immunotoxicity induced by TCDD during pregnancy in the mother and fetus. We observed that RES protected not only normal nonpregnant mice but also pregnant mothers and their fetuses from TCDD-induced thymic atrophy, apoptosis, and alterations in the expression of T-cell receptor and costimulatory molecules as well as T-cell differentiation. In addition, there was significantly reduced expression of CYP1A1 in thymi of both the mother and the fetus when RES was used in vivo post-TCDD exposure. In conclusion, these studies demonstrate that consumption of RES, a natural plant product, during pregnancy, may afford protection to the mother and the fetus from the toxicity induced by environmental pollutants that mediate their effects through activation of aryl hydrocarbon receptor.

Possible involvement of arylhydrocarbon receptor variants in TCDD-induced thymic atrophy and XRE-dependent transcriptional activity in Wistar Hannover GALAS rats

Wistar Hannover Global Alliance for Laboratory Animal Standardization (WH GALAS) rats have been distributed for international standardization of preclinical and toxicological research. Han/Wistar (Kuopio) rats are exceptionally resistant to acute toxicities caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and mediated by the aryl hydrocarbon receptor (AhR), and they have a mutated AhR, named AhR(hw/hw). We found that the WH GALAS rat has either of the three AhR allele, AhR(wt/wt), AhRwt/hw and AhRhw/hw. We administered TCDD (0, 5 and 10 microg/kg) to Long-Evans (L-E) rats having AhR(wt/wt) and two WH GALAS rat strains having either AhR(wt/wt) or AhR(hw/hw), and examined the weights of their body, liver and thymus 168 hr post-administration. WH GALAS AhR(hw/hw) strain was more resistant to TCDD-induced effects on thymus weight than L-E and WH GALAS AhR(wt/wt) strains. In order to study differences in susceptibility of thymic atrophy among the strains, we examined CYP1A1 mRNA and AhR protein levels between L-E and WH GALAS strains. However, no significant difference was observed in the amount of AhR protein or CYP1A1 mRNA in the thymus. Next, we carried out in vitro assays to examine the transactivation activities of AhR variants and found that the AhR deletion variant (AhRdv) transcribed from AhR(hw/hw) significantly enhanced transactivation activity of the synthesized xenobiotic response element. All AhR variants similarly suppressed the growth of Jurkat T cells upon TCDD exposure. This study suggests that WH GALAS rat having different AhR alleles is an interesting experimental animal model but should be utilized with caution for preclinical research on chemicals having AhR agonistic activities.

Enhanced Expression of Plasma Glutathione Peroxidase in the Thymus of Mice Treated with TCDD and Its Implication for TCDD-induced Thymic Atrophy

Enhanced Expression of Plasma Glutathione Peroxidase in the Thymus of Mice Treated with TCDD and Its Implication for TCDD-induced Thymic Atrophy

Treatment of Mice with 2,3,7,8-Tetrachlorodibenzo-p-Dioxin Leads to Aryl Hydrocarbon Receptor-Dependent Nuclear Translocation of NF-κB and Expression of Fas Ligand in Thymic Stromal Cells and Consequent Apoptosis in T Cells

We investigated the role of aryl hydrocarbon receptor (AhR) in the regulation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced apoptosis in thymic T cells. AhR knockout (KO) mice were resistant to TCDD-induced thymic atrophy and apoptosis when compared with the AhR wild-type mice. TCDD triggered the expression of several apoptotic genes, including FasL in AhR wild-type but not AhRKO mice. TCDD-induced increase in FasL was seen only in thymic stromal but not thymic T cells. When TCDD-exposed stromal cells were mixed with untreated thymic T cells, increased apoptosis was detected in T cells that involved Fas-FasL interactions. Thus, apoptosis in T cells was not detected when TCDD-treated stromal cells from FasL-defective or AhRKO mice were mixed with wild-type T cells or when TCDD-exposed wild-type stromal cells were mixed with Fas-deficient T cells. TCDD treatment, in vivo and in vitro, led to colocalization and translocation of NF-kappaB subunits (p50, p65) to the nucleus in stromal but not T cells from AhR wild-type mice. NF-kappaB activation was not observed in stromal cells isolated from TCDD-treated AhRKO mice. Mutations in NF-kappaB-binding sites on the FasL promoter showed that TCDD regulates FasL promoter activity through NF-kappaB. TCDD treatment in vivo caused activation of the death receptor and mitochondrial pathways of apoptosis. Cross-talk between the two pathways was not necessary for apoptosis inasmuch as TCDD-treated Bid KO mice showed thymic atrophy and increased apoptosis, similar to the wild-type mice. These findings demonstrate that AhR regulates FasL and NF-kappaB in stromal cells, which in turn plays a critical role in initiating apoptosis in thymic T cells.

Evidence for induction of apoptosis in T cells from murine fetal thymus following perinatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

Perinatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes thymic atrophy, but the precise mechanism of such toxicity remains unresolved. The current study investigated the role of apoptosis in TCDD-induced thymic involution following perinatal exposure to TCDD. To this end, C57BL/6 pregnant mice were injected intraperitoneally on gestational day (GD) 14 with a single dose of 10 microg/kg TCDD. Analysis of the thymus on GDs 15, 16, 17, and 18, and on postnatal day (PD) 1, showed a remarkable reduction in thymic cellularity 3-7 days post-TCDD exposure. TCDD treatment also caused marked changes in the proportions of T-cell subsets, particularly on GD 17 and GD 18 thymocytes. In vitro culture of thymocytes from mice exposed perinatally to TCDD showed increased apoptosis when compared to the controls, which peaked on day 3 post-TCDD exposure. Triple-color staining showed that TCDD induced apoptosis in all four subpopulations of T cells, with the double-positive T cells undergoing the highest level. Moreover, increased cleavage of caspase-3 was seen when TCDD-exposed GD 17 thymocytes were directly tested. Furthermore, apoptosis-associated phenotypic changes were found in thymocytes of mice perinatally exposed to TCDD, characterized by an increase in expression of CD3, alphabetaTCR, IL-2R, and CD44, and a decrease in CD4, CD8, and J11d markers. Finally, thymocytes from mice exposed perinatally to TCDD showed higher levels of Fas, TRAIL, and DR5 mRNA, but the levels of Bcl-2, Bcl-xL, and Bax were either unaltered or changed moderately. Taken together, these results suggest that TCDD-induced thymic atrophy following perinatal exposure may result, at least in part, from increased apoptosis mediated by death receptor pathway involving Fas, TRAIL, and DR5.

Cell proliferation arrest within intrathymic lymphocyte progenitor cells causes thymic atrophy mediated by the aryl hydrocarbon receptor.

Activation of the aryl hydrocarbon receptor (AHR), a basic helix-loop-helix transcription factor, in lymphocytes by the immunosuppressive environmental contaminant 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD) has been shown to cause thymic atrophy in every species studied. We set out to identify the specific hemopoietic cellular populations in which the AHR was activated to lead to thymic atrophy and to determine the effect of AHR activation in those cellular populations. Initially, we examined whether AHR activation in intrathymic dendritic cells could mediate TCDD-induced thymic atrophy. It was found that thymic atrophy occurred only when the AHR could be activated in the thymocytes but not hemopoietic-derived dendritic cells or other APCs. We next analyzed the effect of TCDD on the proliferation of thymocytes in vivo. There was a significant increase in the percentage of thymocytes in the G(1) phase of the cell cycle and a significant decrease in the percentage of S plus G(2)/M thymocytes, especially in the CD4(-)CD8(-)CD3(-) triple-negative intrathymic progenitor cell population 24 h after exposure to 30 micro g/kg TCDD. Furthermore, by 12 h after exposure to TCDD, we observed approximately 60% reduction of 5-bromo-2'-deoxyuridine incorporation in specific intrathymic progenitor cell populations. This reduction persisted for at least 6 days. These data indicate that intrathymic progenitor cells are direct targets of TCDD in the thymus and suggest that TCDD causes thymic atrophy by reducing entrance into cell cycle in these populations.

T cell-specific disruption of arylhydrocarbon receptor nuclear translocator (Arnt) gene causes resistance to 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced thymic involution.

The arylhydrocarbon receptor nuclear translocator (ARNT) is a member of the basic helix-loop-helix, PER-ARNT-SIM family of heterodimeric transcription factors, and serves as a dimerization partner for arylhydrocarbon receptor (AHR) and hypoxia-inducible factor-1alpha. To assess the function of ARNT in T cells, we disrupted the Arnt gene specifically in T cells of mice by conditional gene targeting using T cell-specific p56(lck)-Cre (Lck-Cre) transgenic Arnt-floxed mice. Thus generated, T cell-specific Arnt-disrupted mice (Lck-Cre;Arnt(flox/Delta) transgenic mice) exhibited complete loss of the expression of ARNT protein only in T cells, and were viable and appeared normal. The Arnt-disrupted T cells in the thymus were phenotypically and histologically normal. The Arnt-deficient T cells in the spleen were capable of responding to TCR stimulation in vitro. However, unlike normal mice in which exposure to the environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an AHR ligand, resulted in thymic involution, the thymus of Lck-Cre;Arnt(flox/Delta) mice were resistant to TCDD treatment in vivo. In contrast, benzo(a)pyrene, another AHR ligand, still caused thymic involution in Lck-Cre;Arnt(flox/Delta) mice. Finally, fetal thymus organ culture using Lck-Cre;Arnt(flox/Delta) and K5-Cre;Arnt(flox/Delta) (epithelial cell-specific Arnt-disrupted mice) showed that thymocytes rather than thymic epithelial cells are predominantly responsible for TCDD-induced thymic atrophy. Our results indicate that ARNT in T lineage cells is essential for TCDD-mediated thymic involution.

2,3,7,8-tetrachlorodibenzo-p-dioxin causes alterations in lymphocyte development and thymic atrophy in hemopoietic chimeras generated from mice deficient in ARNT2.

It is well established that dioxins cause a variety of toxic effects and syndromes including alterations of lymphocyte development. Exposure to the prototypical dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) leads to severe thymic atrophy in all species studied. It has been shown that most of this toxicity is due to TCDD binding to and activating the aryl hydrocarbon receptor (AHR). Upon activation, the AHR enters the nucleus, dimerizes with the AHR nuclear translocator (ARNT), and this heterodimer modulates a number of genes that mediate toxicity. The AHR and ARNT are members of the basic-helix-loop-helix-Per, ARNT, and Sim homology (bHLH-PAS) family of transcription factors. In this study, we wanted to determine if another bHLH-PAS transcription factor, ARNT2, which has high amino acid sequence identity to ARNT and has been shown to dimerize with the TCDD-activated AHR, is involved in mediating TCDD's effect on lymphocyte development. We determined by RT-PCR that ARNT2 is expressed at a low level in whole thymus, thymocytes, and bone marrow lymphocytes. We created hemopoietic chimeras by lethally irradiating C57BL/6 mice and reconstituting them with fetal liver stem cells that either have or are deficient in a portion of chromosome 7 that contains ARNT2. Regardless of whether chimeras possessed or lacked this chromosome fragment, equal sensitivity to TCDD-induced thymic atrophy was observed despite expression of ARNT2 in the thymus. Furthermore, the absence of ARNT2 (or any other genes found on this portion of chromosome 7) did not confer any protection against TCDD-induced alterations in bone marrow B-cell subsets. These data indicate that in this model system the effects of TCDD-induced thymic atrophy and alterations in B-cell maturation are not dependent on an AHR-ARNT2 heterodimer.

Hemopoietic progenitor cells are sensitive targets of 2,3,7,8-tetrachlorodibenzo-p-dioxin in C57BL/6J mice.

Treatment of adult C57BL6J mice with tetrachlorodibenzo-p-dioxin (TCDD) elicits altered bone marrow hemopoietic cellular potentials and markedly reduced T-lymphoid-reconstituting activity. The latter has been hypothesized to play a role in TCDD-induced thymic atrophy. To investigate cellular targets responsible for reduced prothymocyte capacity, bone marrow cells from TCDD-treated C57BL/6J mice were assessed for hemopoietic alterations within the lineage-negative (lin-) compartment by the examination of Sca-1 and c-Kit levels. Lin- hemopoietic cells from C57BL/6J mice, treated with 30 microg/kg of TCDD, were assessed for phenotypic alterations following 24 h through 31 days. The responses of lin- cells to TCDD doses ranging from 0.3 to 30 microg/kg were also assessed at 2 days following TCDD treatment. The data reveal increases in the number of bone marrow lin- Sca-1+ c-Kit+ cells, relative to control, over 24 h through 31 days following treatment, as well as dose-dependent increases in this population when examined at 2 days. Increases in lin- Sca-1+ c-Kit- cells occurred on a more transient basis and were also dependent upon TCDD dose. These data suggest that proliferation and/or differentiation processes of hemopoietic stem cells are affected by TCDD and that these effects contribute to a reduced capacity of bone marrow to generate pro-T lymphocytes.

Overexpression of the Anti-apoptotic Oncogene, bcl-2, in the Thymus Does Not Prevent Thymic Atrophy Induced by Estradiol or 2,3,7,8-Tetrachlorodibenzo-p-Dioxin

Dexamethasone (Dex), estradiol (E2), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) all affect the immune system, causing immunosuppression and thymic atrophy. It is still uncertain how and where these compounds act to induce thymic atrophy. However, it has been suggested that these compounds may have similar actions and targets, i.e., apoptosis of immature thymocytes for Dex and TCDD and preferential targeting of double-positive cells by Dex and E2. Thelckpr-bcl-2 transgenic mouse has been shown to be protected against Dex-induced thymic atrophy. We used this murine model to determine if bcl-2 expression would also protect against E2- and TCDD-induced thymic atrophy. Our results indicate that, although the bcl-2 transgenic (TG+) mice were fully protected from atrophy induced by a single dose of Dex, atrophy was still induced in these mice following treatment with E2 or TCDD. Phenotypic analysis of thymocytes from TG− and TG+ mice also showed distinct consequences of atrophy induced by Dex, E2, and TCDD. Finally, since there are alternative pathways for apoptosis that are bcl-2 independent, both TG− and TG+ thymocytes were examined directly for indications of apoptosis using the TUNEL assay. After TCDD and E2 treatment there were no detectable signs of apoptosis in either TG− or TG+ mice even at early time points and at elevated dose levels. These results indicate that there are distinct mechanisms for the actions of Dex, E2, and TCDD in the thymus and that apoptosis is not a key mechanism of E2- and TCDD-induced thymic atrophy.

Thymic Alterations Induced by 2,3,7,8-Tetrachlorodibenzo-p-Dioxin Are Strictly Dependent on Aryl Hydrocarbon Receptor Activation in Hemopoietic Cells

Abstract2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related congeners affect the immune system, causing immunosuppression and thymic atrophy in a variety of animal species. TCDD is believed to exert its effects primarily through the ligand-activated transcription factor, the aryl hydrocarbon receptor (AhR). Although the AhR is found at high levels in both thymocytes and thymic stroma, it is uncertain in which cells TCDD is activating the AhR to cause alterations in the thymus. Some investigators have suggested that stromal elements, primarily epithelial cells, within the thymus are the primary targets for TCDD. Others have suggested that atrophy is due to a direct effect on thymocytes, either by apoptosis or by altering the development of progenitor cells. By producing chimeric mice with TCDD-responsive (AhR+/+) stromal components and TCDD-unresponsive (AhR−/−) hemopoietic components, or the reverse, we have clarified the role of stromal vs hemopoietic elements in TCDD-induced thymic alterations. Our results show that the targets for TCDD-induced thymic atrophy and phenotypic alterations are strictly in the hemopoietic compartment and that TCDD activation of epithelial cells in the stroma is not required for thymic alterations. Furthermore, changes observed in the putative stem cell populations of these chimeric mice are also dependent on TCDD activation of the AhR in hemopoietic elements.

Aryl-hydrocarbon Receptor-Deficient Mice Are Resistant to 2,3,7,8-Tetrachlorodibenzo-p-dioxin-Induced Toxicity

Acute exposure of mammals to the environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) results in a diverse set of toxicologic and pathologic effects. The mechanism of some of these effects has been studied extensivelyin vitroand correlative studies have indicated the involvement of a transcription factor known as the aryl hydrocarbon receptor (AHR). However, a definitive association of the AHR with TCDD-mediated toxicity has been difficult to establish due to the diversity of effects and the ubiquitous expression of this receptor. In an effort to distinguish AHR-mediated TCDD toxicities from those resulting from alternative pathways, we have made use of the recently described AHR-deficient mouse that was generated by locus-specific homologous recombination in embryonic stem cells. Present studies demonstrate that AHR-deficient mice are relatively unaffected by doses of TCDD (2000 μg/kg) 10-fold higher than that found to induce severe toxic and pathologic effects in littermates expressing a functional AHR. Analyses of liver, thymus, heart, kidney, pancreas, spleen, lymph nodes, and uterus from AHR-deficient mice identified no significant TCDD-induced lesions. The resistance of AHR-deficient mice to TCDD-induced thymic atrophy appears restricted to processes involving AHR since the corticosteroid dexamethasone rapidly and efficiently induced cortical depletion in both AHR-deficient and normal littermate control mice. Taken together these results suggest that the pathological changes induced by TCDD in the liver and thymus are mediated entirely by the AHR. However, it is important to note that at high doses of TCDD, AHR-deficient mice displayed limited vasculitis and scattered single cell necrosis in their lungs and livers, respectively. The mechanism(s) responsible for these apparently receptor-independent processes remain unclear but may involve novel, alternative pathways for TCDD-induced toxicity.

No evidence for emergence of autoreactive V β6 + T cells in Mls-1 a mice following exposure to a thymotoxic dose of 2,3,7,8-tetrachlorodibenzo- p-dioxin

Tolerance to minor lymphocyte stimulatory-1 a (Mls-1 a) antigens is associated with clonal deletion of cells carrying the T cell receptor variable region Vβ6. Thymic epithelial cells may contribute to the intrathymic negative selection of potentially autoreactive Vβ6 + cells. Because 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) acts on thymic epithelial cells, we hypothesized that it may interfere with intrathymic selection processes. In the present study, this was addressed by exposing Mls-1 a DBA/2 mice to a single thymotoxic dose of TCDD. The emergence of Vβ6 + cells in thymus, spleen, and mesenteric lymph nodes was investigated during the subsequent recovery of TCDD-induced thymic atrophy. In addition, the extrathymic differentiation of T lymphocytes in the liver was studied. TCDD exposure resulted in a severe thymic atrophy, and an increase in hepatic mononuclear cells. However, we were not able to demonstrate any emergence of potentially autoreactive mature Vβ6 + T cells, that differentiated either intrathymically or extrathymically, in TCDD-exposed DBA/2 mice.

Dexamethasone, β-Estradiol, and 2,3,7,8-Tetrachlorodibenzo- p-dioxin Elicit Thymic Atrophy through Different Cellular Targets

The effects of single doses of dexamethasone (DEX), β-estradiol-17-valerate (E2), and 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) on the kinetics of thymic atrophy and related bone marrow and thymocyte phenotqpe alterations were examined. The results imply differences in the mechanisms by which these compounds act. Of the three compounds, DEX induced maximal atrophy by 3 days with complete recovery by Day 12. At the point of maximal atrophy, the RAG-1 +TdT +CD4 +8 +3 int thymocyte population was proportionately the most depleted. In contrast, TCDD and E2 caused maximal thymic atrophy by Day 12. E2 treatment, like DEX, resulted in a preferential decrease in the RAG-1 +TdT +CD4 +8 +3 int population, but unlike DEX, this decrease persisted. TCDD-induced thymic atrophy resulted from a proportional loss of all classes of thymocytes. There was no significant relative reduction of TdT +RAG-1 + cells by TCDD in the thymus. A slow and persistent reduction of TdT and RAG-1 in bone marrow by both TCDD and E2 contrasted with the rapid reduction and quick recovery of these markers in marrow from DEX-treated animals. Additional studies showed that only DEX-induced atrophy was accompanied by the induction of thymocyte apoptosis, as detected by multiple nucleosomal length DNA fragments within the first 24 hr. The different kinetics and proportions of subsets in the atrophied thymuses, as well as the distinct patterns of alterations of RAG and TdT expression, and the presence or the absence of apoptosis provide evidence for different mechanisms of thymic atrophy by these agents. The slow induction and longer persistence of thymic atrophy induced by E2 and TCDD, as well as their effects on bone marrow stem cell markers, suggest that bone marrow thymocyte precursors are major targets for these agents.

2,3,7,8-Tetrachlorodibenzo- p-dioxin-induced thymic atrophy and lymphocyte stem cell alterations by mechanisms independent of the estrogen receptor

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) has both agonist and antagonist effects on estrogen-mediated activities and estrogen receptor (ER) levels in epithelial tissues following exposure. We previously demonstrated that TCDD alters bone marrow lymphocyte stem cells, including prothymocytes, as measured by functional assays and alterations in the lymphocyte stem cell-specific markers terminal deoxynucleotidyl transferase (TdT) and recombinase activating gene-1 (RAG-1). We have also shown that 17 β-estradiol valerate (E 2V) affects lymphocyte stem cells by reducing TdT and RAG-1 mRNA. It has been suggested that the effect of TCDD on these lymphocyte stem cells may be mediated directly or indirectly through estrogenic action and/or the ER. Studies were designed to evaluate whether endogenous estrogens or the ER mediate TCDD-elicited bone marrow alterations and thymic atrophy. Ovariectomy did not alter the sensitivity of mice to TCDD-induced thymic atrophy or to a reduction in TdT biosynthesis in bone marrow cells compared with either intact or sham-operated mice. The pure estrogen antagonist ICI 164,384 blocked E 2V-induced uterine hypertrophy, thymic atrophy and reductions in lymphocyte stem cell markers. However, the antiestrogen failed to protect against TCDD-elicited thymic atrophy or bone marrow alterations in intact animals. The results are consistent with the hypothesis that the effects of TCDD on the thymus and/or bone marrow are mediated by mechanisms independent of estrogens or the ER.

Alterations in the cortical thymic epithelium of rats after in vivo exposure to 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD): An (immuno)histological study

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) induces thymic atrophy in rats. The present study was initiated to provide (immuno)histological data on the mechanism of action. Juvenile male Wistar rats were orally intubated once with 50 or 150 μg/kg TCDD. They were euthanized 4 or 10 days thereafter, or were allowed to stay alive until Day 20 or 26. Growth retardation occurred rapidly in all TCDD-treated animals. Lethality was demonstrated within 20–21 days after administration. At Days 4 and 10 after intubation, thymic atrophy was shown by reduction of thymic weight and cortex/medulla ratio. Staining patterns for T-cell markers in the atrophic thymuses coincided with the reduction of cortical areas. There was no evidence indicating that the effects were indirectly caused by stress. TCDD-induced thymic atrophy persisted until Day 26 after administration. Immunohistochemical analysis revealed prominent changes in the cortical thymic epithelium at the 150-μg/kg dose level. First, in the cortex epithelial cell aggregates were observed both at Day 4 and at Day 10 after administration. Apparently, the architecture of the epithelium had changed in these animals. Second, at 10 days after administration epithelial cells were found with the simultaneous expression of markers that in the normal uninvoluted thymus only occur either in the subcapsular/medullary area or in the cortex. This phenotype points to an unusual stage of differentiation. We conclude that TCDD exposure affects the cortical epithelium of the rat thymus at a high dose level. Apparently, it disturbs the epithelial network and interferes with the differentiation of epithelial cells.

Impairment of prothymocyte activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Exposure of experimental animals to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) results in severe thymic atrophy and suppression of cell-mediated and humoral immune functions. However, despite much effort the mechanism by which TCDD produces these responses, particularly thymic atrophy, remains unclear. In this report, we have examined the effect of acute TCDD exposure on lymphocyte stem cells in young adult BALB/c mice to determine whether alterations to events early in T lymphopoiesis contribute to TCDD-induced thymic atrophy. TCDD produced a dose-dependent reduction in thymic weight and cellularity following a single dose of 5 to 120 micrograms TCDD/kg. This thymic atrophy correlated with a dose-dependent suppression of the biosynthesis and mRNA levels of the lymphocyte stem cell-specific DNA polymerase terminal deoxynucleotidyl transferase in bone marrow and thymus. However, the reduction in thymic terminal deoxynucleotidyl nucleotidyl transferase synthesis, on a per cell basis, was less than that observed in bone marrow. Intrathymic CD4/CD8 and IL-2R expression demonstrated only mild alterations after exposure to 30 micrograms TCDD/kg. These data suggest that thymocytes are more refractory to TCDD than are pre-T cells. To assess this possibility directly, bone marrow prothymocytes from TCDD-treated donor mice were examined for their capacity to reconstitute the thymuses of adoptive, irradiated recipients. Our results indicate that prothymocyte activity was severely impaired by TCDD exposure and that this effect occurred at low tissue levels of TCDD. In contrast, we observed no reduction in the number of colony-forming unit-granulocyte macrophage and a moderate decrease in colony-forming unit-spleen. These data suggest that TCDD-induced thymic atrophy is the result, at least in part, of impaired thymic seeding by prothymocytes.

2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) kills glucocorticoid-sensitive thymocytes invivo

The effect of in vivo exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on glucocorticoid- and calcium ionophore-induced DNA fragmentation in rat thymocytes was investigated. TCDD dose-dependently abolished DNA fragmentation in response to both agents after 7 days of exposure. Analysis of the time dependence of the effect revealed that after 1 or 2 days TCDD potentiated DNA fragmentation in untreated and glucocorticoid-treated thymocyte suspensions relative to controls. The DNA fragmentation in untreated thymocyte suspensions from TCDD-treated rats was completely prevented by inhibitors that block glucocorticoid-induced thymocyte suicide. Our results suggest that TCDD-induced thymic atrophy is due to Ca2+-dependent endonuclease activation.

Evidence for direct action of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) on thymic epithelium

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) acts on selected targets within the immune system to produce a characteristic profile of pathologic responses typified by thymic atrophy, suppressed cellular immunity, and inhibition of antibody production to T-lymphocyte-dependent antigens. Studies in inbred mice differing in sensitivity to TCDD indicate that TCDD-induced thymic atrophy is mediated by a receptor protein (designated the Ah receptor). To study the cellular and molecular basis for TCDD-induced thymic atrophy, primary cultures of thymic epithelial (TE) cells were established from C57BL 6 mice, a strain sensitive to TCDD. Treatment of TE monolayers with TCDD (0.1 to 10 n m) resulted in the altered maturation of cocultured syngeneic thymocytes as judged by suppression (40% of control at 10 n m TCDD) of TE-dependent responsiveness of thymocytes to the mitogens concanavalin A and phytohemagglutinin. TE-conditioned medium enhanced the mitogen responsiveness of thymocytes three- to four-fold; however, the enhanced mitogen response mediated by the TE-conditioned medium was not suppressed in thymocytes incubated in medium collected from TCDD-treated cultures or in TE-conditioned medium to which TCDD (10 n m) had been added directly. The suppression of TE-dependent maturation of thymocytes was concentration dependent (EC 50 ∼ 1 n m) and stereospecific, suggesting involvement of the Ah receptor. The Ah receptor in cytosol fractions from cultured TE cells was measured directly and was found to be present at a concentration 3 and 3.5 times greater than that measured in whole thymus and thymocytes, respectively. The results of this study indicate that TCDD can act directly on epithelial target cells in the thymus: one consequence of this action appears to be the altered thymus-dependent maturation of T-lymphocyte precursors, mediated through direct cell-cell contact between thymocytes and TE cells.