Timothy R. Zacharewski

Interaction of PAH-related compounds with the α and β isoforms of the estrogen receptor

The ability of several 4- and 5-ring polycyclic aromatic hydrocarbons (PAHs), heterocyclic PAHs, and their monohydroxy derivatives to interact with the estrogen receptor (ER) alpha and beta isoforms was examined. Only compounds possessing a hydroxyl group were able to compete with 3H-labeled 17β-estradiol (E2) for binding to either a glutathione-S-transferase and human ERα D, E, and F domain fusion protein (GST-hERαdef) or to the full-length human ERβ. Competitive binding was comparable for both isoforms, with IC50 values ranging from 20 to 300 nM (E2 IC50 approximately 3 nM). However, several compounds were able to induce reporter gene expression preferentially through mERβ, using MCF-7 cells transiently transfected with either a Gal4-human ERαdef or Gal4-mouse ERβdef construct, as well as a Gal4-regulated reporter. These data extend the number and type of PAH-related compounds capable of interacting with ERα and ERβ, and provides additional evidence that even though some compounds may possess a similar affinity for both ER isoforms, the capacity for transcriptional activation can still be isoform-specific.

Quantification of rainbow trout (Oncorhynchus mykiss) zona radiata and vitellogenin mRNA levels using real-time PCR after in vivo treatment with estradiol-17β or α-zearalenol

Estrogen receptor-mediated induction of zona radiata (ZR) and vitellogenin (VTG) mRNA and protein in rainbow trout (Oncorhynchus mykiss) was compared to assess their utility as biomarkers for exposure to estrogenic compounds. Partial sequences of rainbow trout ZR and beta-actin were cloned by reverse transcriptase polymerase chain reaction (RT-PCR) using degenerate primers based on conserved regions across a number of species. A 549 bp fragment of the rainbow trout ZR-gene showed a high degree of amino acid sequence identity to that of salmon (77%), winter flounder (64%), carp ZP2 (63%) and medaka (61%) ZR-proteins. The 1020 bp beta-actin fragment was approximately 100% identical to sequences from several species. Real-time PCR was used to quantify the induction of ZR-gene and VTG in rainbow trout liver after in vivo exposure to estradiol-17 beta (E(2)) (0.01, 0.1, 1.0 or 10 mg/kg body weight (bw) fish) or alpha-zearalenol (alpha-ZEA) (0.1, 1.0 or 10 mg/kg bw). Real-time PCR and indirect enzyme-linked immunosorbent assay (ELISA) showed that ZR and VTG were induced in both the liver and the plasma after a single injection of E(2) or alpha-ZEA. ZR was more responsive to low levels of E(2) and alpha-ZEA than VTG, and real-time PCR was shown to be more sensitive than the ELISA. Rainbow trout ZR-gene and proteins provide a sensitive biomarker for assessing estrogenic activity.

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.

Effect of human dietary exposure levels of genistein during gestation and lactation on long-term reproductive development and sperm quality in mice.

The objective of the present study was to determine the long-term reproductive effects of gestational and lactational exposure (0, 0.1, 0.5, 2.5 and 10 mg/kg/day) to genistein on male mice at levels comparable to or greater than human dietary exposures. Testicular growth, sperm count and motility, and sperm fertilizing ability in vitro was assessed in male offspring on postnatal days (PND) 105 and 315. Selected genes were also examined by real-time PCR to determine whether genistein caused changes in gene expression similar to those previously observed with diethylstilbestrol (DES). No significant treatment-related effects on male offspring body weight, anogenital distance, seminal vesicle weight or testis weight were observed. There were also no significant effects on sperm count, the percent of motile sperm or the number of motile sperm at any age. The in vitro fertilizing ability of epididymal sperm was increased significantly in the high-dose group approximately 17% (P < 0.001) on PND 105 and 315. The results indicate that developmental exposure of mice to genistein at human exposure levels does not induce adverse effects on sperm quality or changes in testicular gene expression similar to DES.

Distribution of androgen receptor mRNA expression and immunoreactivity in the brain of the green anole lizard.

Male courtship and copulation are androgen dependent in the green anole lizard, and female receptivity can be facilitated by testosterone. However, only a few, and relatively large, regions in the brain have been implicated in the control of these behaviours. In situ hybridization and immunohistochemistry were therefore used to determine in detail where androgens are likely to act in the brains of breeding males and females. A 697‐bp fragment of the anole androgen receptor (AR) was cloned from total RNA isolated from the kidney, which contains the highly androgen‐sensitive renal sex segment. The cloned fragment spanned part of the C, the entire D, and part of the E domains, and shared a high degree of similarity with the AR of various species. 35S‐labelled antisense and sense probes were generated from the 697‐bp fragment for use in in situ hybridization, and the AR antibody PG‐21 was used for immunohistochemistry. Both sexes consistently had AR mRNA expression and immunoreactivity in areas associated with vertebrate reproductive behaviours and in motor areas of the brainstem. Interestingly, the PG‐21 antibody produced labelling in both the nucleus and cytoplasm, including neuronal processes. The distribution of mRNA and immunoreactivity were comparable in males and females, and the amount of labelling was generally similar, although slightly greater in females. The expression pattern of AR in this species supports the idea that distribution is highly conserved among vertebrates, but that it probably does not dictate behavioural differences between the sexes in anoles.

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.

Empirical Bayes Gene Screening Tool for Time-Course or Dose-Response Microarray Data

Abstract An efficient method to reduce the dimensionality of microarray gene expression data from thousands or tens of thousands of cDNA clones down to a subset of the most differentially expressed cDNA clones is essential in order to simplify the massive amount of data generated from microarray experiments. An extension to the methods of Efron et al. [Efron, B., Tibshirani, R., Storey, J., Tusher, V. (2001). Empirical Bayes analysis of a microarray experiment. J. Am. Statist. Assoc. 96:1151–1160] is applied to a differential time-course experiment to determine a subset of cDNAs that have the largest probability of being differentially expressed with respect to treatment conditions across a set of unequally spaced time points. The proposed extension, which is advocated to be a screening tool, allows for inference across a continuous variable in addition to incorporating a more complex experimental design and allowing for multiple design replications. With the current data the focus is on a time-course experiment; however, the proposed methods can easily be implemented on a dose–response experiment, or any other microarray experiment that contains a continuous variable of interest. The proposed empirical Bayes gene-screening tool is compared with the Efron et al. (2001) method in addition to an adjusted model-based t-value using a time-course data set where the toxicological effect of a specific mixture of chemicals is being studied.

Investigation of the Mode of Action Underlying the Tumorigenic Response Induced in B6C3F1 Mice Exposed Orally to Hexavalent Chromium

Chronic ingestion of high concentrations of hexavalent chromium [Cr(VI)] in drinking water induces intestinal tumors in mice. To investigate the mode of action (MOA) underlying these tumors, a 90-day drinking water study was conducted using similar exposure conditions as in a previous cancer bioassay, as well as lower (heretofore unexamined) drinking water concentrations. Tissue samples were collected in mice exposed for 7 or 90 days and subjected to histopathological, biochemical, toxicogenomic, and toxicokinetic analyses. Described herein are the results of toxicokinetic, biochemical, and pathological findings. Following 90 days of exposure to 0.3–520 mg/l of sodium dichromate dihydrate (SDD), total chromium concentrations in the duodenum were significantly elevated at ≥ 14 mg/l. At these concentrations, significant decreases in the reduced-to-oxidized glutathione ratio (GSH/GSSG) were observed. Beginning at 60 mg/l, intestinal lesions were observed including villous cytoplasmic vacuolization. Atrophy, apoptosis, and crypt hyperplasia were evident at ≥ 170 mg/l. Protein carbonyls were elevated at concentrations ≥ 4 mg/l SDD, whereas oxidative DNA damage, as assessed by 8-hydroxydeoxyguanosine, was not increased in any treatment group. Significant decreases in the GSH/GSSG ratio and similar histopathological lesions as observed in the duodenum were also observed in the jejunum following 90 days of exposure. Cytokine levels (e.g., interleukin-1β) were generally depressed or unaltered at the termination of the study. Overall, the data suggest that Cr(VI) in drinking water can induce oxidative stress, villous cytotoxicity, and crypt hyperplasia in the mouse intestine and may underlie the MOA of intestinal carcinogenesis in mice.

Effects of gestational and lactational exposure to Aroclor 1242 on sperm quality and in vitro fertility in early adult and middle-aged mice.

The objective of this study was to examine the effects of gestational and lactational exposure to Aroclor 1242 (0, 10, 25, 50, and 100 mg/kg-bw) on male fertility. Doses were administered to C57BL6 female mice orally every two days from two weeks before mating, during mating, and through gestation until postnatal day 21. Male B6D2F1 offspring were examined for anogenital distance, organ development, epididymal sperm count, sperm motility, and in vitro fertility at 16 and 45 weeks of age. Stomach samples of pups nursing from PCB-treated mothers in the 50 mg/kg dose group were analyzed for PCBs and chlorobiphenylols by high resolution gas chromatography coupled with low resolution mass spectrometry. It was estimated that the nursing pups were exposed to 0.2, 0.6, 1.2, and 2.4 mg/kg/day total PCBs in the 10, 25, 50, and 100 mg/kg dose groups, respectively. This exposure level approaches the maximum FDA recommended levels for PCBs in food and breast milk. The composition of the PCBs in the stomach samples was different from the parent mixture, as there was a higher proportion of heavily chlorinated congeners, as well as chlorobiphenylols. Anogenital distance at weaning, and liver, thymus, and testes weight at 16 and 45 weeks of age were not affected by PCB exposure. Epididymal sperm velocity and linearity were significantly increased in the 25 mg/kg dose group at 16 weeks of age. Sperm count was increased by 36% in this dose group (P = 0.06). By 45 weeks of age, average sperm count in this dose group was similar to that of controls. With the exception of the 50 mg/kg dose group at 16 weeks of age, sperm fertilizing ability in vitro was significantly decreased in all PCB-exposed groups at 16 and 45 weeks of age. These results suggest that fertility in the adult mouse is susceptible to developmental exposure to Aroclor 1242 and is independent of testis weight or epididymal sperm count.

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.