Tim Zacharewski's research while affiliated with Michigan State University and other places

Publications (129)

Article
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental contaminant that induces diverse biological and toxic effects, including reprogramming intermediate metabolism, mediated by the aryl hydrocarbon receptor (AHR). However, the specific reprogramming effects of TCDD are unclear. Here, we performed targeted LC-MS analysis of hepat...
Preprint
2,3,7,8-Tetrachlorodibenzo- p -dioxin (TCDD) dose-dependently induces the development of hepatic fat accumulation and inflammation with fibrosis in mice initially in the portal region. Conversely, differential gene and protein expression is first detected in the central region. To further investigate cell-specific and spatially resolved dose-depend...
Article
Full-text available
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor known for mediating the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. Although the canonical mechanism of AhR activation involves heterodimerization with the aryl hydrocarbon receptor nuclear translocator, other transcriptional regulators...
Article
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The application of single-cell RNA sequencing (scRNAseq) for the evaluation of chemicals, drugs, and food contaminants presents the opportunity to consider cellular heterogeneity in pharmacological and toxicological responses. Current differential gene expression analysis (DGEA) methods focus primarily on two group comparisons, not multi-group dose...
Preprint
Gut dysbiosis with disrupted enterohepatic bile acid metabolism is commonly associated with non-alcoholic fatty liver disease (NAFLD) and recapitulated in a NAFLD-phenotype elicited by 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD) in mice. TCDD induces hepatic fat accumulation and increases levels of secondary bile acids including taurolithocholic a...
Preprint
Full-text available
The application of single-cell RNA sequencing (scRNAseq) for the evaluation of chemicals, drugs, and food contaminants presents the opportunity to consider cellular heterogeneity in pharmacological and toxicological responses. Current differential gene expression analysis (DGEA) methods focus primarily on two group comparisons, not multi-group dose...
Preprint
Full-text available
2,3,7,8 –Tetrachlorodibenzo – p –dioxin (TCDD) is a persistent environmental contaminant and the prototypical ligand for the aryl hydrocarbon receptor (AhR). AhR mediates the effects of TCDD and related compounds, including the reprograming of intermediate metabolism. Untargeted metabolomics analysis of hepatic extracts prepared from mice orally ga...
Article
Full-text available
The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor in the PAS superfamily of environmental sensors that is linked to several metabolic diseases, including non-alcoholic fatty liver disease (NAFLD). Much remains unknown regarding the impact of genetic variation in AHR-driven disease, as past studies have focused on a smal...
Preprint
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a persistent environmental contaminant, induces steatosis by increasing hepatic uptake of dietary and mobilized peripheral fats, inhibiting lipoprotein export, and repressing β-oxidation. In this study, the mechanism of β-oxidation inhibition was investigated by testing the hypothesis that TCDD dose-depen...
Article
Full-text available
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a persistent environmental contaminant, induces steatosis that can progress to steatohepatitis with fibrosis, pathologies that parallel stages in the development of non-alcoholic fatty liver disease (NAFLD). Coincidently, one carbon metabolism (OCM) gene expression and metabolites are often altered during...
Article
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Background and Aims Characterization of cell specific transcriptional responses to hepatotoxicants is lost in the averages of bulk RNA-sequencing (RNA-seq). Single-cell/nuclei RNA-seq technologies enable the transcriptomes of individual cell (sub)types to be assessed within the context of in vivo models. Methods Single-nuclei RNA-sequencing (snSeq...
Article
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent aryl hydrocarbon receptor (AhR) agonist that elicits a broad spectrum of dose-dependent hepatic effects including lipid accumulation, inflammation, and fibrosis. To determine the role of inflammatory lipid mediators in TCDD-mediated hepatotoxicity, eicosanoid metabolism was investigated. Female...
Preprint
Full-text available
Cell-specific transcriptional responses are lost in the averages of bulk RNA sequencing. We performed single nuclei RNA sequencing (snSeq) on frozen liver samples from male C57BL/6 mice in response to 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD). Approximately 19,907 hepatic genes were detected across 16,015 sequenced nuclei from control and treate...
Article
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Acetamide (CAS 60-35-5) is detected in common foods. Chronic rodent bioassays led to its classification as a group 2B possible human carcinogen due to the induction of liver tumors in rats. We used a toxicogenomics approach in Wistar rats gavaged daily for 7 or 28 days at doses of 300 to 1500 mg/kg/day (mkd) to determine a point of departure (POD)...
Data
Generation of liver-specific gene set. Microarray datasets for basal gene expression for 96 different male mouse tissues/cell types were obtained from Lattin et al. [34]. The difference between the microarray signal in the liver and the maximum fluorescent signal on a per gene basis in all other tissues/cells was calculated (ΔSignal). A gene was co...
Article
Full-text available
The aryl hydrocarbon receptor (AhR) is a highly conserved transcription factor that mediates a broad spectrum of species-, strain-, sex-, age-, tissue-, and cell-specific responses elicited by structurally diverse ligands including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Dose-dependent effects on liver-specific and sexually dimorphic gene expre...
Data
Comparison of liver-specific and sexually dimorphic gene sets. (DOCX)
Data
Gene expression changes of sexually dimorphic genes. (XLSX)
Data
Chemiluminescence traces of Wes protein assays. Data was collected and analyzed as described in materials & methods. (DOCX)
Data
Gene expression changes in TCDD treated mice for 181 liver-specific genes. (XLSX)
Article
We have previously shown that in response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-elicited NAFLD progression, central carbon, glutaminolysis and serine/folate metabolism are reprogrammed to support NADPH production and ROS defenses. To further investigate underlying dose-dependent responses associated with TCDD-induced fibrosis, female C57BL/...
Article
Full-text available
Dose-dependent differential gene expression provides critical information required for regulatory decision-making. The lower costs associated with RNA-Seq have made it the preferred technology for transcriptomic analysis. However, concordance between RNA-Seq and microarray analyses in dose response studies has not been adequately vetted. We compare...
Article
Full-text available
Toxic equivalency factors (TEFs) are assigned to dioxin-like chemicals based on relative potency (REP) values of individual adaptive and toxic responses compared to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Agilent 4x44K oligonucleotide microarrays were used to examine the hepatic gene expression potency of 2,3,7,8-tetrachlorodibenzofuran (TCDF),...
Chapter
The goals of this chapter are to 1) introduce genomics, transcriptomics, proteomics, and metabolomics technologies; 2) describe the advantages and challenges associated with these approaches compared to traditional methodologies, particularly from the perspective of cross-species extrapolation within human and environmental toxicology; and 3) ident...
Article
Refinement of in vitro systems for predictive toxicology is important in order to develop high-throughput early toxicity screening assays and to minimize animal testing studies. This study assesses the ability of mouse Hepa-1c1c7 hepatoma cell model under differing culture conditions to predict in vivo estrogen-induced hepatic gene expression chang...
Article
The estrogenic activity of 17beta-estradiol (E2), alpha-zearalenol (alpha-ZEA), genistein (GEN), and 4-t-octylphenol (4-t-OP) was investigated using Xenopus laevis-based assays. All test compounds competed with [3H]E2 for binding to a recombinant Xenopus estrogen receptor (xER) with the following relative affinities: E2 > alpha-ZEA > 4-t-OP > GEN....
Article
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Hepa-1c1c7 wild-type and benzo[a]pyrene-resistant derived mutant cell lines have been used to elucidate pathways and mechanisms involving the aryl hydrocarbon receptor (AhR). However, there has been little focus on other biological processes which may differ between the isolated lines. In this study, mouse cDNA microarrays representing 4858 genes w...
Article
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Motivation: An important underlying assumption of any experiment is that the experimental subjects are similar across levels of the treatment variable, so that changes in the response variable can be attributed to exposure to the treatment under study. This assumption is often not valid in the analysis of a microarray experiment due to systematic...
Article
Full-text available
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 t...
Chapter
The statistics associated with drug development are astonishing. Historically, only one in 5000-10,000 chemicals screened successfully reach the market, only 30% of marketed drugs produce sufficient revenue to recover research and development costs, new drug development requires 12 to 15 years and a $500-800 million investment, and 30-50% of drug c...
Article
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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. [Efro...
Article
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Comparative approaches were used to identify human, mouse and rat dioxin response elements (DREs) in genomic sequences unambiguously assigned to a nucleotide RefSeq accession number. A total of 13 bona fide DREs, all including the substitution intolerant core sequence (GCGTG) and adjacent variable sequences, were used to establish a position weight...
Chapter
Transcription factors (TFs) are responsible for regulating the temporal and tissue specific expression of the genome. Identifying the complete battery of genes that are targeted by individual TFs, how these target genes interact within a cellular network, and the role these networks play in various disease states is essential to pharmacology and to...
Article
Estrogen induction of uterine wet weight provides an excellent model to investigate relationships between changes in global gene expression and well-characterized physiological responses. In this study, time course microarray GeneChip data were analyzed using a novel approach to identify temporal changes in uterine gene expression following treatme...
Article
The estrogen receptor acts primarily as an estrogen-inducible transcription factor in target organs, however, until recently the identification of individual responsive transcripts has been cumbersome. In the present study, oligonucleotide GeneChip microarrays were used to describe and analyze the levels of approximately 6500 transcripts in the ute...
Article
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...
Article
Recent investigations have demonstrated that polyphenolic catechins inhibit breast cancer cell proliferation and tumor growth. However, the ER-mediated effects of the three predominant catechins (EGCG, ECG, and EGC) have not been extensively examined in vitro or in vivo. Therefore, EGCG, ECG, and EGC were examined for their ability to compete with...
Article
The ability of 14 structurally diverse estrogenic compounds to induce reporter gene expression mediated by estrogen receptors (ERs) from different species was examined. MCF-7 cells were transiently transfected with a Gal4-regulated luciferase reporter gene (17m5-G-Luc) and Gal4-ER chimeric receptors containing the D, E and F domains of the human (G...
Article
Full-text available
The objective of the study was to determine the long-term effects of gestational and lactational exposure to diethylstilbestrol (DES; 0, 0.1, 1, and 10 microg/kg maternal body weight) on mouse testicular growth, epididymal sperm count, in vitro fertilizing ability, and testicular gene expression using cDNA microarrays and real-time PCR in mice on p...
Article
The objective of the study was to determine the effect of in utero and lactational exposure to genistein (0, 0.1, 0.5, 2.5 and 10 mg/kg/day) on mammary gland morphology in female B6D2F1 mice at levels comparable to or greater than human exposures. The effect of diethylstilbestrol (DES; 0, 0.1, 1, 10 microg/kg/day) on the mammary gland was also exam...
Article
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Here we describe an automated and customizable program to correct, filter and normalize raw microarray data captured using GenePix, a commonly used microarray image analysis application. Files can be processed individually or in batch mode for increased throughput. User defined inputs specify the stringency of data filtering and the method and cond...
Article
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Bioinformatics, or in silico biology, is a rapidly growing field that encompasses the theory and application of computational approaches to model, predict, and explain biological function at the molecular level. This information rich field requires new skills and new understanding of genome-scale studies in order to take advantage of the rapidly in...
Article
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...
Article
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 17beta-estradiol (E2) for binding to either a glutathione...
Article
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...
Article
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-...
Article
RNA and protein expression profiling technologies have revolutionized how toxicologists can study the molecular basis of adverse effects of chemicals and drugs. It is expected that these new technologies will afford efficient and high-throughput means to delineate mechanisms of action and predict toxicity of unknown agents. To reach these goals, a...
Article
The estrogenic activities of bisphenol A (BPA) and its major metabolite BPA glucuronide (BPA-G) were assessed in a number of in vitro and in vivo assays. BPA competed with [3H]-17beta-estradiol (E2) for binding to mouse uterine cytosol ER, a glutathione S-transferase (GST)-human ER D, E, and F domain fusion protein (GST-hERalphadef) and full-length...
Article
Full-text available
The estrogenic activities of benzo[a]pyrene (B[a]P) and 10 metabolites (1, 3-, 7-, and 9-hydroxy-B[a]P; 4,5-, 7,8-, and 9,10-dihydrodihydroxy-B[a]P; and 1,6-, 3,6-, and 6,12-B[a]P-dione) were investigated. In vitro, B[a]P did not displace tritiated 17beta-estradiol ([3H]E2) from either a bacterially expressed fusion protein consisting of glutathion...
Article
Human sex hormone-binding globulin (SHBG) binds sex steroids with high affinity. In plasma, the number of SHBG steroid-binding sites far exceeds the molar concentrations of sex steroids, and will accommodate other ligands such as phytoestrogens and fatty acids. We have therefore developed a screening assay to identify ligands for SHBG, which exist...
Article
The study investigated the ability of 34 natural and synthetic chemicals to compete with [3H]17β-estradiol (E2) for binding to bacterially expressed glutathione-S-transferase (GST)-estrogen receptors (ER) fusion proteins from five different species. Fusion proteins consisted of the ER D, E and F domains of human alpha (GST-hERαdef), mouse alpha (GS...
Article
Estrogen receptor (ER) transactivation assays were initially designed to study endogenous mechanisms of steroid hormone action, but more recently have been used to assess industrial chemicals for potential estrogenic activity. Given the diverse spectrum of physicochemical properties of these chemicals, we examined the effects of pH (a weak organic...
Article
Full-text available
Estrogen receptor (ER) transactivation assays were initially designed to study endogenous mechanisms of steroid hormone action, but more recently have been used to assess industrial chemicals for potential estrogenic activity. Given the diverse spectrum of physicochemical properties of these chemicals, we examined the effects of pH (a weak organic...
Article
Full-text available
A human breast cancer cell line, MCF-7, transiently transfected with a chimeric estrogen receptor (Gal4-HEG0) and a luciferase reporter plasmid (17m5-G-Luc), was used to investigate the estrogenic activity of benzo[a]pyrene (B[a]P), a prototypical polyaromatic hydrocarbon (PAH). B[a]P at concentrations > or = 1 microM produced responses comparable...
Article
We evaluated the feasibility of incorporating an exogenous metabolic activating system into an estrogen receptor-alpha transactivation assay. 17beta-estradiol (E2), and the proestrogenic pesticide methoxychlor (MXC) were evaluated for activity in the presence and absence of Aroclor-1254 induced rat liver S-9 fractions. Both E2 and MXC responded con...
Article
A coal-tar creosote was examined for estrogen receptor (ER)–and aryl hydrocarbon receptor (AhR)–mediated activity using a battery of mechanistically based assays. In vitro, creosote was found to bind to the mouse ER, bind to the human sex hormone–binding globulin, and elicit partial agonist activity in reporter gene assays in transiently transfecte...
Article
Full-text available
A comparative study was undertaken to assess the ability of 44 polychlorinated biphenyls (PCBs), 9 hydroxylated PCBs (HO-PCBs), and 8 aroclors at concentrations ranging from 1 nM to 10 microM to compete with [3H]17beta-estradiol (E2) for binding to bacterially expressed fusion proteins using a semi-high throughput competitive-binding assay. The fus...
Article
A coal-tar creosote was examined for estrogen receptor (ER)–and aryl hydrocarbon receptor (AhR)–mediated activity using a battery of mechanistically based assays. In vitro, creosote was found to bind to the mouse ER, bind to the human sex hormone–binding globulin, and elicit partial agonist activity in reporter gene assays in transiently transfecte...
Article
Full-text available
The estrogenic activities of eight phthalate esters (i.e., di-2-ethylhexyl, di-n-butyl (DBP), butylbenzyl (BBP), di-hexyl (DHP), diiso-heptyl, di-n-octyl, diiso-nonyl, diiso-decyl) were investigated in vitro using estrogen receptor (ER) competitive ligand-binding and mammalian- and yeast-based gene expression assays. In vivo, their effects on uteri...