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1.  Development of a stable cell line with an intact PGC-1alpha/ERRalpha axis for screening environmental chemicals 
The estrogen-related receptor α (ERRα) and the peroxisome proliferator-activated receptor γ (PPARγ) coactivator 1α (PGC-1α) play critical roles in the control of several physiological functions, including the regulation of genes involved in energy homeostasis. However, little is known about the ability of environmental chemicals to disrupt or modulate this important bioenergetics pathway in humans. The goal of this study was to develop a cell-based assay system with an intact PGC-1α/ERRα axis that could be used as a screening assay for detecting such chemicals. To this end, we successfully generated several stable cell lines expressing PGC-1α and showed that the reporter driven by the native ERRα hormone response unit (AAB-Luc) is active in these cell lines and that the activation is PGC-1α-dependent. Furthermore, we show that this activation can be blocked by the ERRα selective inverse agonist, XCT790. In addition, we find that genistein and bisphenol A further stimulate the reporter activity, while kaempferol has minimal effect. These cell lines will be useful for identifying environmental chemicals that modulate this important pathway.
PMCID: PMC3967403  PMID: 24457025
2.  The p53 transcriptional target gene wnt7b contributes to NGF-inducible neurite outgrowth in neuronal PC12 cells 
Differentiation of PC12 cells by nerve growth factor (NGF) is characterized by changes in signal transduction pathways leading to growth arrest and neurite extension. The transcription factor p53, involved in regulating cell cycle and apoptosis, is also activated during PC12 differentiation and contributes to each of these processes but the mechanisms are incompletely understood. NGF signaling stabilizes p53 protein expression which enables its transcriptional regulation of target genes, including the newly identified target, wnt7b, a member of the wnt family of secreted morphogens. We tested the hypothesis that wnt7b expression is a factor in NGF-dependent neurite outgrowth of differentiating PC12 cells. Wnt7b transcript and protein levels are increased following NGF treatment in a p53-dependent manner, as demonstrated by a reduction in wnt7b protein levels following stable shRNA-mediated silencing of p53. In addition, overexpressed human tp53 was capable of inducing marked wnt7b expression in neuronal PC12 cells but tp53 overexpression did not elevate wnt7b levels in several tested human tumor cell lines. Ectopic wnt7b overexpression was sufficient to rescue neurite outgrowth in NGF-treated p53-silenced PC12 cells which could be blocked by JNK inhibition with SP600125 and did not involve β-catenin nuclear translocation. Addition of sFRP1 to differentiation medium inhibited wnt7b-dependent phosphorylation of JNK, demonstrating that wnt7b is secreted and signals through a JNK-dependent mechanism in PC12 cells. We further identify an NGF-inducible subset of wnt receptors that likely supports wnt7b-mediated neurite extension in PC12 cells. In conclusion, wnt7b is a novel p53-regulated neuritogenic factor in PC12 cells that in conjunction with NGF-regulated Fzd expression is involved in p53-dependent neurite outgrowth through noncanonical JNK signaling.
PMCID: PMC2562925  PMID: 18177422
wnt7b; wnt; p53; PC12; NGF; neurite; JNK; Fzd
3.  Nerve growth factor potentiates p53 DNA binding but inhibits nitric oxide-induced apoptosis in neuronal PC12 cells. 
Neurochemical research  2007;32(9):1573-1585.
NGF is recognized for its role in neuronal differentiation and maintenance. Differentiation of PC12 cells by NGF involves p53, a transcription factor that controls growth arrest and apoptosis. We investigated NGF influence over p53 activity during NO-induced apoptosis by sodium nitroprusside in differentiated and mitotic PC12 cells. NGF-differentiation produced increased p53 levels, nuclear localization and sequence-specific DNA binding. Apoptosis in mitotic cells also produced these events but the accompanying activation of caspases 1–10 and mitochondrial depolarization were inhibited during NGF differentiation and could be reversed in p53-silenced cells. Transcriptional regulation of PUMA and survivin expression were not inhibited by NGF, although NO-induced mitochondrial depolarization was dependent upon de novo gene transcription and only occurred in mitotic cells. We conclude that NGF mediates prosurvival signaling by increasing factors such as Bcl-2 and p21Waf1/Cip1 without altering p53 transcriptional activity and prevents mitochondrial depolarization and caspase activation to inhibit apoptosis.
PMCID: PMC2231119  PMID: 17592775
NO; PC12; mitochondria; differentiation; NGF; p53
4.  Alterations in the Rat Serum Proteome During Liver Injury from Acetaminophen Exposure 
Changes in the serum proteome were identified during early, fulminant and recovery phases of liver injury from acetaminophen in the rat. Male F344 rats received a single, non-injury dose or a high, injury-producing dose of acetaminophen for evaluation at 6 hr to 120 hr. Two-dimensional gel electrophoresis of immunodepleted serum separated about 800 stained proteins per sample from which differentially expressed proteins were identified by mass spectrometry. Serum ALT/AST levels and histopathology revealed the greatest liver damage at 24 and 48 hr after high dose acetaminophen corresponding to the time of greatest serum protein alterations. After 24 hr, 68 serum proteins were significantly altered of which 23 proteins were increased by >5 fold and 20 proteins were newly present compared to controls. Only minimal changes in serum proteins were noted at the low dose without any histopathology. Of the 54 total protein isoforms identified by mass spectrometry, gene ontology processes for 38 unique serum proteins revealed involvement of acute phase response, coagulation, protein degradation, intermediary metabolism and various carrier proteins. Elevated serum TNFα from 24 to 48 hr suggested a mild inflammatory response accompanied by increased antioxidant capability demonstrated by increased serum catalase activity. Antibody array and ELISA analyses also showed elevation in the chemokine, MCP-1, and the metalloprotease inhibitor, TIMP-1, during this same period of liver injury. This study demonstrates that serum proteome alterations likely reflect both liver damage and a concerted, complex response of the body for organ repair and recovery during acute hepatic injury.
PMCID: PMC1892200  PMID: 16687475
ACTH18-39, adrenocorticotropic hormone fragment 18-39; ALT, alanine aminotransferase; ANIT, α-naphthylisothiocyanate; ARP3, actin related protein-3; AST, aspartate aminotransferase; BSA, bovine serum albumin; CHAPS, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulphonate; CINC-2 and -3, Cytokine induced neutrophil chemoattractant-2 and -3; EDTA, ethylenediamine-tetraacetic acid; EGTA, ethylene glycol-bis(ß-aminoethylether)-N,N,N’,N’-tetra-acetic acid; ECL, enhanced chemiluminescence; ECM, extracellular matrix; EGF-1, epidermal growth factor-1; ELISA, enzyme-linked immunosorbent assay; EST, expressed sequence tag; GST, glutathione S-transferase; GM-CSF, granulocyte-macrophage colony stimulating factor; GRP, glucose responsive protein; HGF, hepatic growth factor; HEPES, N-2-Hydroxyethyl-piperazine-N'-2-ethane-sulfonic acid; 4-HPPD, 4- hydroxyphenylpyruvate dioxygenase; HRP, horseradish peroxidase; ICAT, isotope coded affinity tags; IPG, immobilized pH gradients; IEF, isoelectric focusing; LC, liquid chromatography; LIX, LPS-induced C-X-C chemokine; MALDI, matrix assisted laser desorption ionization; MCP1, monocyte chemoattractant protein-1; MS/MS, tandem mass spectrometry; Mr, relative molecular mass; MS, mass spectrometry; MSN, master spot number; PAGE, polyacrylamide gel electrophoresis; pI, isoelectric point; qRT-PCR, quantitative reverse transcriptase-polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SUMO1, small ubiquitin-like modifier-1; TIMP-1, tissue inhibitor of metalloproteinases-1; TNFα, tumor necrosis factor-alpha
6.  Bisphenol A affects androgen receptor function via multiple mechanisms 
Chemico-biological interactions  2013;203(3):556-564.
Bisphenol A (BPA), is a well-known endocrine disruptor compound (EDC) that affects the normal development and function of the female and male reproductive system, however the mechanisms of action remain unclear. To investigate the molecular mechanisms of how BPA may affect ten different nuclear receptors, stable cell lines containing individual nuclear receptor ligand binding domain (LBD)-linked to the β-Gal reporter were examined by a quantitative high throughput screening (qHTS) format in the Tox21 Screening Program of the NIH. The results showed that two receptors, estrogen receptor alpha (ERα) and androgen receptor (AR), are affected by BPA in opposite direction. To confirm the observed effects of BPA on ERα and AR, we performed transient transfection experiments with full-length receptors and their corresponding response elements linked to luciferase reporters. We also included in this study two BPA analogs, bisphenol AF (BPAF) and bisphenol S (BPS). As seen in African green monkey kidney CV1 cells, the present study confirmed that BPA and BPAF act as ERα agonists (half maximal effective concentration EC50 of 10-100 nM) and as AR antagonists (half maximal inhibitory concentration IC50 of 1-2 μM). Both BPA and BPAF antagonized AR function via competitive inhibition of the action of synthetic androgen R1881. BPS with lower estrogenic activity (EC50 of 2.2 μM), did not compete with R1881 for AR binding, when tested at 30 μM. Finally, the effects of BPA were also evaluated in a nuclear translocation assays using EGPF-tagged receptors. Similar to 17β-estradiol (E2) which was used as control, BPA was able to enhance ERα nuclear foci formation but at a 100-fold higher concentration. Although BPA was able to bind AR, the nuclear translocation was reduced. Furthermore, BPA was unable to induce functional foci in the nuclei and is consistent with the transient transfection study that BPA is unable to activate AR.
PMCID: PMC3722857  PMID: 23562765
Bisphenol A and related compounds; androgen receptor; qHTS; transfection; imaging analysis
7.  p53 integrates host defense and cell fate during bacterial pneumonia 
p53 deletion augments neutrophil-mediated bacterial clearance in the lung at the expense of tissue homeostasis, leading to increased mortality.
Cancer and infection are predominant causes of human mortality and derive, respectively, from inadequate genomic and host defenses against environmental agents. The transcription factor p53 plays a central role in human tumor suppression. Despite its expression in immune cells and broad responsiveness to stressors, it is virtually unknown whether p53 regulates host defense against infection. We report that the lungs of naive p53−/− mice display genome-wide induction of NF-κB response element–enriched proinflammatory genes, suggestive of type 1 immune priming. p53-null and p53 inhibitor–treated mice clear Gram-negative and -positive bacteria more effectively than controls after intrapulmonary infection. This is caused, at least in part, by cytokines produced by an expanded population of apoptosis-resistant, TLR-hyperresponsive alveolar macrophages that enhance airway neutrophilia. p53−/− neutrophils, in turn, display heightened phagocytosis, Nox-dependent oxidant generation, degranulation, and bacterial killing. p53 inhibition boosts bacterial killing by mouse neutrophils and oxidant generation by human neutrophils. Despite enhanced bacterial clearance, infected p53−/− mice suffer increased mortality associated with aggravated lung injury. p53 thus modulates host defense through regulating microbicidal function and fate of phagocytes, revealing a fundamental link between defense of genome and host during environmental insult.
PMCID: PMC3646498  PMID: 23630228
8.  RNA-Seq Profiling Reveals Novel Hepatic Gene Expression Pattern in Aflatoxin B1 Treated Rats 
PLoS ONE  2013;8(4):e61768.
Deep sequencing was used to investigate the subchronic effects of 1 ppm aflatoxin B1 (AFB1), a potent hepatocarcinogen, on the male rat liver transcriptome prior to onset of histopathological lesions or tumors. We hypothesized RNA-Seq would reveal more differentially expressed genes (DEG) than microarray analysis, including low copy and novel transcripts related to AFB1’s carcinogenic activity compared to feed controls (CTRL). Paired-end reads were mapped to the rat genome (Rn4) with TopHat and further analyzed by DESeq and Cufflinks-Cuffdiff pipelines to identify differentially expressed transcripts, new exons and unannotated transcripts. PCA and cluster analysis of DEGs showed clear separation between AFB1 and CTRL treatments and concordance among group replicates. qPCR of eight high and medium DEGs and three low DEGs showed good comparability among RNA-Seq and microarray transcripts. DESeq analysis identified 1,026 differentially expressed transcripts at greater than two-fold change (p<0.005) compared to 626 transcripts by microarray due to base pair resolution of transcripts by RNA-Seq, probe placement within transcripts or an absence of probes to detect novel transcripts, splice variants and exons. Pathway analysis among DEGs revealed signaling of Ahr, Nrf2, GSH, xenobiotic, cell cycle, extracellular matrix, and cell differentiation networks consistent with pathways leading to AFB1 carcinogenesis, including almost 200 upregulated transcripts controlled by E2f1-related pathways related to kinetochore structure, mitotic spindle assembly and tissue remodeling. We report 49 novel, differentially-expressed transcripts including confirmation by PCR-cloning of two unique, unannotated, hepatic AFB1-responsive transcripts (HAfT’s) on chromosomes 1.q55 and 15.q11, overexpressed by 10 to 25-fold. Several potentially novel exons were found and exon refinements were made including AFB1 exon-specific induction of homologous family members, Ugt1a6 and Ugt1a7c. We find the rat transcriptome contains many previously unidentified, AFB1-responsive exons and transcripts supporting RNA-Seq’s capabilities to provide new insights into AFB1-mediated gene expression leading to hepatocellular carcinoma.
PMCID: PMC3632591  PMID: 23630614
9.  Testing an Aflatoxin B1 Gene Signature in Rat Archival Tissues 
Chemical Research in Toxicology  2012;25(5):1132-1144.
Archival tissues from laboratory studies represent a unique opportunity to explore the relationship between genomic changes and agent-induced disease. In this study, we evaluated the applicability of qPCR for detecting genomic changes in formalin-fixed, paraffin-embedded (FFPE) tissues by determining if a subset of 14 genes from a 90-gene signature derived from microarray data and associated with eventual tumor development could be detected in archival liver, kidney, and lung of rats exposed to aflatoxin B1 (AFB1) for 90 days in feed at 1 ppm. These tissues originated from the same rats used in the microarray study. The 14 genes evaluated were Adam8, Cdh13, Ddit4l, Mybl2, Akr7a3, Akr7a2, Fhit, Wwox, Abcb1b, Abcc3, Cxcl1, Gsta5, Grin2c and C8orf46 homolog. The qPCR FFPE liver results were compared to the original liver microarray data and to qPCR results using RNA from fresh frozen liver. Archival liver paraffin blocks yielded 30 to 50 μg of degraded RNA that ranged in size from 0.1 to 4 kB. qPCR results from FFPE and fresh frozen liver samples were positively correlated (p≤0.05) by regression analysis and showed good agreement in direction and proportion of change with microarray data for 11 of 14 genes. All 14 transcripts could be amplified from FFPE kidney RNA except the glutamate receptor gene Grin2c; however, only Abcb1b was significantly upregulated from control. Abundant constitutive transcripts, S18 and β-actin, could be amplified from lung FFPE samples, but the narrow RNA size range (25–500 bp length) prevented consistent detection of target transcripts. Overall, a discrete gene signature derived from prior transcript profiling and representing cell cycle progression, DNA damage response, and xenosensor and detoxication pathways was successfully applied to archival liver and kidney by qPCR and indicated that gene expression changes in response to subchronic AFB1 exposure occurred predominantly in liver, the primary target for AFB1-induced tumors. We conclude that an evaluation of gene signatures in archival tissues can be an important toxicological tool for evaluating critical molecular events associated with chemical exposures.
PMCID: PMC3358548  PMID: 22545673
10.  Myeloid Differentiation Primary Response Protein 88 Couples Reverse Cholesterol Transport to Inflammation 
Cell metabolism  2010;11(6):493-502.
Crosstalk exists in mammalian cells between cholesterol trafficking and innate immunity signaling. Apolipoprotein A-I (apoA-I), a serum apolipoprotein that induces anti-atherogenic efflux of macrophage cholesterol, is widely described as anti-inflammatory because it neutralizes bacterial lipopolysaccharide. Conversely, lipopolysaccharide-induced inflammation is pro-atherogenic. However, whether innate immunity plays an endogenous, physiological role in host cholesterol homeostasis in the absence of infection is undetermined. We report that apoA-I signals in the macrophage through Toll like Receptor (TLR)2, TLR4, and CD14, utilizing Myeloid Differentiation Primary Response Protein 88 (MyD88)-dependent and –independent pathways, to activate nuclear factor-κB and induce cytokines. MyD88 plays a critical role in reverse cholesterol transport in vitro and in vivo, in part through promoting ATP-Binding Cassette A1 transporter upregulation. Taken together, this work identifies apoA-I as an endogenous stimulus of innate immunity that couples cholesterol trafficking to inflammation through MyD88, and identifies innate immunity as a physiologic signal in cholesterol homeostasis.
PMCID: PMC3091482  PMID: 20519121
11.  The role of toxicoproteomics in assessing organ specific toxicity 
EXS  2009;99:367-400.
Aims of this chapter on the role of toxicoproteomics in assessing organ-specific toxicity are to define the field of toxicoproteomics, describe its development among global technologies, and show potential uses in experimental toxicological research, preclinical testing and mechanistic biological research. Disciplines within proteomics deployed in preclinical research are described as Tier I analysis, involving global protein mapping and protein profiling for differential expression, and Tier II proteomic analysis, including global methods for description of function, structure, interactions and post-translational modification of proteins. Proteomic platforms used in toxicoproteomics research are briefly reviewed. Preclinical toxicoproteomic studies with model liver and kidney toxicants are critically assessed for their contributions toward understanding pathophysiology and in biomarker discovery. Toxicoproteomics research conducted in other organs and tissues are briefly discussed as well. The final section suggests several key developments involving new approaches and research focus areas for the field of toxicoproteomics as a new tool for toxicological pathology.
PMCID: PMC2853963  PMID: 19157068
12.  Gene expression response in target organ and whole blood varies as a function of target organ injury phenotype 
Genome Biology  2008;9(6):R100.
Histopathology, clinical chemistry, hematology and gene expression data were collected from the rat liver and blood after treatment with eight known hepatotoxins.
This report details the standardized experimental design and the different data streams that were collected (histopathology, clinical chemistry, hematology and gene expression from the target tissue (liver) and a bio-available tissue (blood)) after treatment with eight known hepatotoxicants (at multiple time points and doses with multiple biological replicates). The results of the study demonstrate the classification of histopathological differences, likely reflecting differences in mechanisms of cell-specific toxicity, using either liver tissue or blood transcriptomic data.
PMCID: PMC2481421  PMID: 18570634
13.  NGF-mediated transcriptional targets of p53 in PC12 neuronal differentiation 
BMC Genomics  2007;8:139.
p53 is recognized as a critical regulator of the cell cycle and apoptosis. Mounting evidence also suggests a role for p53 in differentiation of cells including neuronal precursors. We studied the transcriptional role of p53 during nerve growth factor-induced differentiation of the PC12 line into neuron-like cells. We hypothesized that p53 contributed to PC12 differentiation through the regulation of gene targets distinct from its known transcriptional targets for apoptosis or DNA repair.
Using a genome-wide chromatin immunoprecipitation cloning technique, we identified and validated 14 novel p53-regulated genes following NGF treatment. The data show p53 protein was transcriptionally activated and contributed to NGF-mediated neurite outgrowth during differentiation of PC12 cells. Furthermore, we describe stimulus-specific regulation of a subset of these target genes by p53. The most salient differentiation-relevant target genes included wnt7b involved in dendritic extension and the tfcp2l4/grhl3 grainyhead homolog implicated in ectodermal development. Additional targets included brk, sdk2, sesn3, txnl2, dusp5, pon3, lect1, pkcbpb15 and other genes.
Within the PC12 neuronal context, putative p53-occupied genomic loci spanned the entire Rattus norvegicus genome upon NGF treatment. We conclude that receptor-mediated p53 transcriptional activity is involved in PC12 differentiation and may suggest a contributory role for p53 in neuronal development.
PMCID: PMC1894799  PMID: 17540029
14.  Global Gene Expression Associated with Hepatocarcinogenesis in Adult Male Mice Induced by in Utero Arsenic Exposure 
Environmental Health Perspectives  2005;114(3):404-411.
Our previous work has shown that exposure to inorganic arsenic in utero produces hepatocellular carcinoma (HCC) in adult male mice. To explore further the molecular mechanisms of transplacental arsenic hepatocarcinogenesis, we conducted a second arsenic transplacental carcinogenesis study and used a genomewide microarray to profile arsenic-induced aberrant gene expression more extensively. Briefly, pregnant C3H mice were given drinking water containing 85 ppm arsenic as sodium arsenite or unaltered water from days 8 to 18 of gestation. The incidence of HCC in adult male offspring was increased 4-fold and tumor multiplicity 3-fold after transplacental arsenic exposure. Samples of normal liver and liver tumors were taken at autopsy for genomic analysis. Arsenic exposure in utero resulted in significant alterations (p < 0.001) in the expression of 2,010 genes in arsenic-exposed liver samples and in the expression of 2,540 genes in arsenic-induced HCC. Ingenuity Pathway Analysis revealed that significant alterations in gene expression occurred in a number of biological networks, and Myc plays a critical role in one of the primary networks. Real-time reverse transcriptase–polymerase chain reaction and Western blot analysis of selected genes/proteins showed > 90% concordance. Arsenic-altered gene expression included activation of oncogenes and HCC biomarkers, and increased expression of cell proliferation–related genes, stress proteins, and insulin-like growth factors and genes involved in cell–cell communications. Liver feminization was evidenced by increased expression of estrogen-linked genes and altered expression of genes that encode gender-related metabolic enzymes. These novel findings are in agreement with the biology and histology of arsenic-induced HCC, thereby indicating that multiple genetic events are associated with transplacental arsenic hepatocarcinogenesis.
PMCID: PMC1392235  PMID: 16507464
Agilent mouse oligo 22K microarray; arsenic; hepatocellular carcinoma; real-time RT-PCR; transplacental exposure; Western blotting

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