Darrell R. Boverhof

Dioxin Induces an Estrogen-Like, Estrogen Receptor-Dependent Gene Expression Response in the Murine Uterus

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a ubiquitous environmental contaminant that elicits a broad range of toxicities in a tissue-, sex-, age-, and species-specific manner, including alterations in estrogen signaling. Many, if not all, of these effects involve changes in gene expression mediated via the activation of the aryl hydrocarbon receptor (AhR), a ligand activated transcription factor. Recent data indicate that TCDD may also elicit AhR-mediated estrogenic activity through interactions with the estrogen receptor (ER). In an effort to further characterize the estrogenic activity of TCDD, a comprehensive time-course analysis of uterine gene expression was conducted using ovariectomized C57BL/6 mice. Comparison of the temporal uterine transcriptional response to TCDD with that of ethynyl estradiol (EE) revealed a large proportion of the TCDD-mediated gene expression changes were also responsive to EE. Furthermore, pretreatment of mice with the pure ER antagonist ICI 182 780 (faslodex) inhibited gene expression responses to both EE and TCDD, providing additional evidence that these transcriptional responses involve the ER.

Protocols for the assurance of microarray data quality and process control

Microarrays represent a powerful technology that provides the ability to simultaneously measure the expression of thousands of genes. However, it is a multi-step process with numerous potential sources of variation that can compromise data analysis and interpretation if left uncontrolled, necessitating the development of quality control protocols to ensure assay consistency and high-quality data. In response to emerging standards, such as the minimum information about a microarray experiment standard, tools are required to ascertain the quality and reproducibility of results within and across studies. To this end, an intralaboratory quality control protocol for two color, spotted microarrays was developed using cDNA microarrays from in vivo and in vitro dose-response and time-course studies. The protocol combines: (i) diagnostic plots monitoring the degree of feature saturation, global feature and background intensities, and feature misalignments with (ii) plots monitoring the intensity distributions within arrays with (iii) a support vector machine (SVM) model. The protocol is applicable to any laboratory with sufficient datasets to establish historical high- and low-quality data.

In vivo – in vitro toxicogenomic comparison of TCDD-elicited gene expression in Hepa1c1c7 mouse hepatoma cells and C57BL/6 hepatic tissue

BackgroundIn vitro systems have inherent limitations in their ability to model whole organism gene responses, which must be identified and appropriately considered when developing predictive biomarkers of in vivo toxicity. Systematic comparison of in vitro and in vivo temporal gene expression profiles were conducted to assess the ability of Hepa1c1c7 mouse hepatoma cells to model hepatic responses in C57BL/6 mice following treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).ResultsGene expression analysis and functional gene annotation indicate that Hepa1c1c7 cells appropriately modeled the induction of xenobiotic metabolism genes in vivo. However, responses associated with cell cycle progression and proliferation were unique to Hepa1c1c7 cells, consistent with the cell cycle arrest effects of TCDD on rapidly dividing cells. In contrast, lipid metabolism and immune responses, representative of whole organism effects in vivo, were not replicated in Hepa1c1c7 cells.ConclusionThese results identified inherent differences in TCDD-mediated gene expression responses between these models and highlighted the limitations of in vitro systems in modeling whole organism responses, and additionally identified potential predictive biomarkers of toxicity.

Inhibition of estrogen-mediated uterine gene expression responses by dioxin.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exhibits antiestrogenic properties, including the inhibition of estrogen-induced uterine growth and proliferation. The inhibition of estrogen-mediated gene expression through ER/AhR cross-talk has been proposed as a plausible mechanism; however, only a limited number of inhibited responses have been investigated that are unlikely to fully account for the antiuterotrophic effects of TCDD. Therefore, the effects of TCDD on ethynyl estradiol (EE)-mediated uterine gene expression were investigated using cDNA microarrays with complementary physiological and histological phenotypic anchoring. Mice were gavaged with vehicle, 3 daily doses of 10 μg/kg EE, a single dose of 30 μg/kg TCDD, or a combination of EE plus TCDD and sacrificed after 4, 12, 24, and 72 h. TCDD cotreatment inhibited EE-induced uterine wet weight by 37, 23, and 45% at 12, 24, and 72 h, respectively. TCDD cotreatment also reduced EE-mediated stromal edema, hypertrophy, and hyperplasia and induced marked luminal epithelial cell apoptosis. A 2 × 2 factorial microarray design was used to identify EE- and TCDD-specific differential gene expression responses as well as their interactive effects. Only 133 of the 2753 EE-mediated differentially expressed genes were significantly modulated by TCDD cotreatment, indicating a gene-specific inhibitory response. The EE-mediated induction of many genes, including trefoil factor 1 and keratin 14, were inhibited by greater than 90% by TCDD. Functional annotation of inhibited responses was associated with cell proliferation, water and ion transport, and maintenance of cellular structure and integrity. These inhibited responses correlate with the observed histological alterations and may contribute to the antiuterotrophic effects of TCDD.

Comparative Temporal Toxicogenomic Analysis of TCDD- and TCDF-Mediated Hepatic Effects in Immature Female C57BL/6 Mice

Temporal analyses were performed on hepatic tissue from immature female C57BL/6 mice in order to compare the gene expression profiles for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 2,3,7,8-tetrachlorodibzofuran (TCDF). Time course studies conducted with a single oral dose of 300 microg/kg TCDF or 30 microg/kg TCDD were used to compare differential gene expression on complementary DNA microarrays containing 13,361 features, representing 8194 genes at 2, 4, 8, 12, 24, 72, 120, and 168 h. One hundred and ninety-five genes were identified as differentially regulated by TCDF, of which 116 genes were in common with TCDD, with 109 exhibiting comparable expression profiles (correlation coefficients > 0.3). In general, TCDF was less effective in eliciting hepatic vacuolization, and differential gene expression compared with TCDD when given at an equipotent dose based on a toxic equivalence factor (TEF) of 0.1 for TCDF, especially 72-h postadministration. For example, the induction of Cyp1a1 messenger RNA by TCDF was less when compared TCDD. Moreover, TCDF induced less severe hepatocyte cytoplasmic vacuolization consistent with lower lipid accumulations which significantly subsided by 120 and 168 h when compared with TCDD. TCDF-elicited responses correlated with their hepatic tissue levels which gradually decreased between 18 and 168 h. Although both compounds elicited comparable gene expression profiles, especially at early time points, the TCDF responses were generally weaker. Collectively, these results suggest that the weaker TCDF responses could be attributed to differences in pharmacokinetics. However, more comprehensive dose-response studies are required at optimal times for each end point of interest in order to investigate the effect of pharmacokinetic differences on relative potencies that are important in establishing TEFs.

Toxicogenomic evaluation of long-term hepatic effects of TCDD in immature, ovariectomized C57BL/6 mice.

Acute exposure to hepatotoxic doses of 2,3,7,8-tetrachloro- dibenzo-p-dioxin (TCDD) in mice is characterized by differential gene expression that can be phenotypically anchored to elevated levels of serum alanine aminotransferase, increased relative liver weights, hepatic steatosis, inflammation, and hepatocellular necrosis. Unlike most studies that focus on acute exposure effects, this study evaluated the long-term effects of a single oral gavage of 30 μg/kg TCDD at 1, 4, 12, 24, 36, and 72 weeks postdose in ovariectomized C57BL/6 mice. Hepatic TCDD levels were almost completely eliminated by 24 weeks with a calculated half-life of 12 days. Hepatic gene expression analysis identified 395 unique differentially expressed genes between 1 and 12 weeks that decreased to ≤ 8 by 72 weeks, consistent with the minimal hepatic TCDD levels. Hepatic vacuolization, characteristic of short-term exposure, subsided by 4 weeks. Similarly, TCDD-elicited hepatic necrosis and inflammation dissipated by 1 week. Collectively, these results suggest that TCDD-elicited histologic and gene expression responses can be correlated to elevated hepatic TCDD levels, which, once eliminated, elicit minimal hepatic gene expression and histologic alterations.