A 50-gene biomarker identifies estrogen receptor-modulating chemicals in a microarray compendium.

High throughput transcriptomics (HTTr) profiling has the potential to rapidly and comprehensively identify molecular targets of environmental chemicals that can be linked to adverse outcomes. We describe here the construction and characterization of a 50-gene expression biomarker designed to identify estrogen receptor (ER) active chemicals in HTTr datasets. Using microarray comparisons, the genes in the biomarker were identified as those that exhibited consistent directional changes when ER was activated (4 ER agonists; 4 ESR1 gene constitutively active mutants) and opposite directional changes when ER was suppressed (4 antagonist treatments; 4 ESR1 knockdown experiments). The biomarker was evaluated as a predictive tool using the Running Fisher algorithm by comparison to annotated gene expression microarray datasets including those evaluating the transcriptional effects of hormones and chemicals in MCF-7 cells. Depending on the reference dataset used, the biomarker had a predictive accuracy for activation of up to 96%. To demonstrate applicability for HTTr data analysis, the biomarker was used to identify ER activators in a set of 15 chemicals that are considered potential bisphenol A (BPA) alternatives examined at up to 10 concentrations in MCF-7 cells and analyzed by full-genome TempO-Seq. Using benchmark dose (BMD) modeling, the biomarker genes stratified the ER potency of BPA alternatives consistent with previous studies. These results demonstrate that the ER biomarker can be used to accurately identify ER activators in transcript profile data derived from MCF-7 cells.

Differential genomic effects of four nano-sized and one micro-sized CeO2 particles on HepG2 cells.

The objective of this research was to perform a genomics study of five cerium oxide particles, 4 nano and one micrometer-sized particles which have been studied previously by our group with respect to cytotoxicity, biochemistry and metabolomics. Human liver carcinoma HepG2 cells were exposed to between 0.3 to 300 ug/ml of CeO2 particles for 72 hours and then total RNA was harvested. Fatty acid accumulation was observed with W4, X5, Z7 and less with Q but not Y6. The gene expression changes in the fatty acid metabolism genes correlated the fatty acid accumulation we detected in the prior metabolomics study for the CeO2 particles named W4, Y6, Z7 and Q, but not for X5. In particular, the observed genomics effects on fatty acid uptake and fatty acid oxidation offer a possible explanation of why many CeO2 particles increase cellular free fatty acid concentrations in HepG2 cells. The major genomic changes observed in this study were sirtuin, ubiquitination signaling pathways, NRF2-mediated stress response and mitochondrial dysfunction. The sirtuin pathway was affected by many CeO2 particle treatments. Sirtuin signaling itself is sensitive to oxidative stress state of the cells and may be an important contributor in CeO2 particle induced fatty acid accumulation. Ubiquitination pathway regulates many protein functions in the cells, including sirtuin signaling, NRF2 mediated stress, and mitochondrial dysfunction pathways. NRF2-mediated stress response and mitochondrial were reported to be altered in many nanoparticles treated cells. All these pathways may contribute to the fatty acid accumulation in the CeO2 particle treated cells.

Determinants of gene expression in the human liver: Impact of aging and sex on xenobiotic metabolism.

There is a need to characterize the potential susceptibility of older adults to toxicity from environmental chemical exposures. Liver xenobiotic metabolizing enzymes (XMEs) play important roles in detoxifying and eliminating xenobiotics. We examined global gene expression in the livers of young (21-45 years) and old (69+ years) men and women. Differentially expressed genes (DEG) were identified using two-way ANOVA (p ≤ 0.05). We identified 1437 and 1670 DEGs between young and old groups in men and women, respectively. Only a minor number of the total number of genes overlapped (146 genes). Aging increased or decreased pathways involved in inflammation and intermediary metabolism, respectively. Aging led to numerous changes in the expression of XME genes or genes known to control their expression (~90 genes). Out of 10 cytochrome P450s activities examined, there were increased activities of CYP1A2 and CYP2C9 enzymes in the old groups. We also identified sex-dependent genes that were more numerous in the young group (1065) than in the old group (202) and included changes in XMEs. These studies indicate that the livers from aging humans when compared to younger adults exhibit changes in XMEs that may lead to differences in the metabolism of xenobiotics.

Effects of Silver Nanoparticles and Silver Nitrate on mRNA and microRNA Expression in Human Hepatocellular Carcinoma Cells (HepG2).

In order to understand toxicity of nano silver, human hepatocellular carcinoma (HepG2) cells were treated either with silver nitrate (AgNO3) or with nano silver capped with glutathione (Ag-S) at various concentration. Differentially expressed genelists for mRNA and microRNA were obtained through Illumina RNA sequencing and DEseq data analyses. Both treatments showed non-linear dose response relationships for mRNA and microRNA. Gene expression analysis showed signaling pathways common to both nano Ag-S and AgNO3, such as cell cycle regulation, DNA damage response and cancer related pathways. But, nano Ag-S caused signaling pathway changes that were not altered by AgNO3 such as NRF2-mediated oxidative stress response inflammation, cell membrane signaling, and cell proliferation. Nano Ag-S also affected p53 signaling, survival, apoptosis, tissue repair, lipid synthesis, angiogenesis, liver fibrosis and tumor development. Several of the pathways affected by nano Ag-S are hypothesized as major contributors to nanotoxicity. MicroRNA target filter analysis revealed additional affected pathways that were not reflected in the mRNA expression response alone, including DNA damage signaling, genomic stability, ROS, cell cycle, ubiquitination, DNA methylation, cell proliferation and fibrosis for AgNO3; and cell cycle regulation, P53 signaling, cell proliferation, survival, apoptosis, tissue repair and so on for nano Ag-S. These pathways may be mediated by microRNA repression of protein translation.Our study clearly showed that the addition of microRNA profiling increased the numbers of signaling pathways discovered that affected by the treatments on HepG2 cells and gave US a better picture of the effects of these reagents in the cells.

Peripheral metabolic effects of ozone exposure in healthy and diabetic rats on normal or high-cholesterol diet.

Epidemiological studies show that individuals with underlying diabetes and diet-associated ailments are more susceptible than healthy individuals to adverse health effects of air pollution. Exposure to air pollutants can induce metabolic stress and increase cardiometabolic disease risk. Using male Wistar and Wistar-derived Goto-Kakizaki (GK) rats, which exhibit a non-obese type-2 diabetes phenotype, we investigated whether two key metabolic stressors, type-2 diabetes and a high-cholesterol atherogenic diet, exacerbate ozone-induced metabolic effects. Rats were fed a normal control diet (ND) or high-cholesterol diet (HCD) for 12 weeks and then exposed to filtered air or 1.0-ppm ozone (6 h/day) for 1 or 2 days. Metabolic responses were analyzed at the end of each day and after an 18-h recovery period following the 2-day exposure. In GK rats, baseline hyperglycemia and glucose intolerance were exacerbated by HCD vs. ND and by ozone vs. air. HCD also resulted in higher insulin in ozone-exposed GK rats and circulating lipase, aspartate transaminase, and alanine transaminase in all groups (Wistar>GK). Histopathological effects induced by HCD in the liver, which included macrovesicular vacuolation and hepatocellular necrosis, were more severe in Wistar vs. GK rats. Liver gene expression in Wistar and GK rats fed ND showed numerous strain differences, including evidence of increased lipid metabolizing activity and ozone-induced alterations in glucose and lipid transporters, specifically in GK rats. Collectively, these findings indicate that peripheral metabolic alterations induced by diabetes and high-cholesterol diet can enhance susceptibility to the metabolic effects of inhaled pollutants.

Diets enriched with coconut, fish, or olive oil modify peripheral metabolic effects of ozone in rats.

Dietary factors may modulate metabolic effects of air pollutant exposures. We hypothesized that diets enriched with coconut oil (CO), fish oil (FO), or olive oil (OO) would alter ozone-induced metabolic responses. Male Wistar-Kyoto rats (1-month-old) were fed normal diet (ND), or CO-, FO-, or OO-enriched diets. After eight weeks, animals were exposed to air or 0.8 ppm ozone, 4 h/day for 2 days. Relative to ND, CO- and OO-enriched diet increased body fat, serum triglycerides, cholesterols, and leptin, while all supplements increased liver lipid staining (OO > FO > CO). FO increased n-3, OO increased n-6/n-9, and all supplements increased saturated fatty-acids. Ozone increased total cholesterol, low-density lipoprotein, branched-chain amino acids (BCAA), induced hyperglycemia, glucose intolerance, and changed gene expression involved in energy metabolism in adipose and muscle tissue in rats fed ND. Ozone-induced glucose intolerance was exacerbated by OO-enriched diet. Ozone increased leptin in CO- and FO-enriched groups; however, BCAA increases were blunted by FO and OO. Ozone-induced inhibition of liver cholesterol biosynthesis genes in ND-fed rats was not evident in enriched dietary groups; however, genes involved in energy metabolism and glucose transport were increased in rats fed FO and OO-enriched diet. FO- and OO-enriched diets blunted ozone-induced inhibition of genes involved in adipose tissue glucose uptake and cholesterol synthesis, but exacerbated genes involved in adipose lipolysis. Ozone-induced decreases in muscle energy metabolism genes were similar in all dietary groups. In conclusion, CO-, FO-, and OO-enriched diets modified ozone-induced metabolic changes in a diet-specific manner, which could contribute to altered peripheral energy homeostasis.

Identification of novel activators of the metal responsive transcription factor (MTF-1) using a gene expression biomarker in a microarray compendium.

Environmental exposure to metals is known to cause a number of human toxicities including cancer. Metal-responsive transcription factor 1 (MTF-1) is an important component of metal regulation systems in mammalian cells. Here, we describe a novel method to identify chemicals that activate MTF-1 based on microarray profiling data. MTF-1 biomarker genes were identified that exhibited consistent, robust expression across 10 microarray comparisons examining the effects of metals (zinc, nickel, lead, arsenic, mercury, and silver) on gene expression in human cells. A subset of the resulting 81 biomarker genes was shown to be altered by knockdown of the MTF1 gene including metallothionein family members and a zinc transporter. The ability to correctly identify treatment conditions that activate MTF-1 was determined by comparing the biomarker to microarray comparisons from cells exposed to reference metal activators of MTF-1 using the rank-based Running Fisher algorithm. The balanced accuracy for prediction was 93%. The biomarker was then used to identify organic chemicals that activate MTF-1 from a compendium of 11 725 human gene expression comparisons representing 2582 chemicals. There were 700 chemicals identified that included those known to interact with cellular metals, such as clioquinol and disulfiram, as well as a set of novel chemicals. All nine of the novel chemicals selected for validation were confirmed to activate MTF-1 biomarker genes in MCF-7 cells and to lesser extents in MTF1-null cells by qPCR and targeted RNA-Seq. Overall, our work demonstrates that the biomarker for MTF-1 coupled with the Running Fisher test is a reliable strategy to identify novel chemical modulators of metal homeostasis using gene expression profiling.

Differential Effects of Nano TiO2 and CeO2 on Normal Human Lung Epithelial Cells In Vitro.

Nano-TiO2 and nano-CeO2 are among the most widely used engineered nanoparticles (NPs). We investigated a variety of endpoints to assess the toxicity of eight of these NPs to induce potentially adverse health effects in an In Vitro human respiratory epithelial cell model. These endpoints include cytotoxicity, reactive oxygen species (ROS)/reactive nitrogen species (RNS) production, 8-hydroxy-2_-deoxyguanosine (8-oxo-dG), endogenous DNA adducts, Apurinic/apyrimidinic (AP) sites, 4-Hrdoxynonenal (4-HNE) protein adducts, Malondialdehyde (MDA) protein adducts, and genomics analysis on altered signaling pathways. Our results indicated that cytotoxicity assays are relatively insensitive, and we detected changes in other endpoints at concentrations much lower than those inducing cytotoxicity. Among the ROS-related endpoints, 8-oxo-dG is relatively more sensitive than other assays, and nano-TiO2 induced more 8-oxo-dG formation than nano-CeO2. Finally, there are many signaling pathways changes at concentrations at which no cytotoxicity was observed. These alterations in signaling pathways correlated well with In Vitro toxicity that was observed at higher concentrations, and with in vivo adverse outcome pathways caused by nano-TiO2 and nano-CeO2 in experimental animals.

Activation of Nrf2 in the liver is associated with stress resistance mediated by suppression of the growth hormone-regulated STAT5b transcription factor.

The transcription factor Nrf2 (encoded by Nfe2l2) induces expression of numerous detoxifying and antioxidant genes in response to oxidative stress. The cytoplasmic protein Keap1 interacts with and represses Nrf2 function. Computational approaches were developed to identify factors that modulate Nrf2 in a mouse liver gene expression compendium. Forty-eight Nrf2 biomarker genes were identified using profiles from the livers of mice in which Nrf2 was activated genetically in Keap1-null mice or chemically by a potent activator of Nrf2 signaling. The rank-based Running Fisher statistical test was used to determine the correlation between the Nrf2 biomarker genes and a test set of 81 profiles with known Nrf2 activation status demonstrating a balanced accuracy of 96%. For a large number of factors examined in the compendium, we found consistent relationships between activation of Nrf2 and feminization of the liver transcriptome through suppression of the male-specific growth hormone (GH)-regulated transcription factor STAT5b. The livers of female mice exhibited higher Nrf2 activation than male mice in untreated or chemical-treated conditions. In male mice, Nrf2 was activated by treatment with ethinyl estradiol, whereas in female mice, Nrf2 was suppressed by treatment with testosterone. Nrf2 was activated in 5 models of disrupted GH signaling containing mutations in Pit1, Prop1, Ghrh, Ghrhr, and Ghr. Out of 59 chemical treatments that activated Nrf2, 36 exhibited STAT5b suppression in the male liver. The Nrf2-STAT5b coupling was absent in in vitro comparisons of chemical treatments. Treatment of male and female mice with 11 chemicals that induce oxidative stress led to activation of Nrf2 to greater extents in females than males. The enhanced basal and inducible levels of Nrf2 activation in females relative to males provides a molecular explanation for the greater resistance often seen in females vs. males to age-dependent diseases and chemical-induced toxicity.

Metabolic Disruption Early in Life is Associated With Latent Carcinogenic Activity of Dichloroacetic Acid in Mice.

Early-life environmental factors can influence later-life susceptibility to cancer. Recent evidence suggests that metabolic pathways may mediate this type of latency effect. Previously, we reported that short-term exposure to dichloroacetic acid (DCA) increased liver cancer in mice 84 weeks after exposure was stopped. Here, we evaluated time course dynamics for key events related to this effect. This study followed a stop-exposure design in which 28-day-old male B6C3F1 mice were given the following treatments in drinking water for up to 93 weeks: deionized water (dH2O, control); 3.5 g/l DCA continuously; or 3.5 g/l DCA for 4-52 weeks followed by dH2O. Effects were evaluated at eight interim time points. A short-term biomarker study was used to evaluate DCA effects at 6, 15, and 30 days. Liver tumor incidence was higher in all DCA treatment groups, including carcinomas in 82% of mice previously treated with DCA for only 4 weeks. Direct effects of DCA in the short-term study included decreased liver cell proliferation and marked mRNA changes related to mitochondrial dysfunction and altered cell metabolism. However, all observed short-term effects of DCA were ultimately reversible, and prior DCA treatment did not affect liver cell proliferation, apoptosis, necrosis, or DNA sequence variants with age. Key intermediate events resulting from transient DCA exposure do not fit classical cytotoxic, mitogenic, or genotoxic modes of action for carcinogenesis, suggesting a distinct mechanism associated with early-life metabolic disruption.

Latent carcinogenicity of early-life exposure to dichloroacetic acid in mice.

Environmental exposures occurring early in life may have an important influence on cancer risk later in life. Here, we investigated carryover effects of dichloroacetic acid (DCA), a small molecule analog of pyruvate with metabolic programming properties, on age-related incidence of liver cancer. The study followed a stop-exposure/promotion design in which 4-week-old male and female B6C3F1 mice received the following treatments: deionized water alone (dH2O, control); dH2O with 0.06% phenobarbital (PB), a mouse liver tumor promoter; or DCA (1.0, 2.0 or 3.5g/l) for 10 weeks followed by dH2O or PB (n = 20-30/group/sex). Pathology and molecular assessments were performed at 98 weeks of age. In the absence of PB, early-life exposure to DCA increased the incidence and number of hepatocellular tumors in male and female mice compared with controls. Significant dose trends were observed in both sexes. At the high dose level, 10 weeks of prior DCA treatment induced comparable effects (≥85% tumor incidence and number) to those seen after continuous lifetime exposure. Prior DCA treatment did not enhance or inhibit the carcinogenic effects of PB, induce persistent liver cytotoxicity or preneoplastic changes on histopathology or alter DNA sequence variant profiles within liver tumors compared with controls. Distinct changes in liver messenger RNA and micro RNA profiles associated with prior DCA treatment were not apparent at 98 weeks. Our findings demonstrate that early-life exposure to DCA may be as carcinogenic as life-long exposures, potentially via epigenetic-mediated effects related to cellular metabolism.

Inhaled ozone (O3)-induces changes in serum metabolomic and liver transcriptomic profiles in rats.

Air pollution has been linked to increased incidence of diabetes. Recently, we showed that ozone (O3) induces glucose intolerance, and increases serum leptin and epinephrine in Brown Norway rats. In this study, we hypothesized that O3 exposure will cause systemic changes in metabolic homeostasis and that serum metabolomic and liver transcriptomic profiling will provide mechanistic insights. In the first experiment, male Wistar Kyoto (WKY) rats were exposed to filtered air (FA) or O3 at 0.25, 0.50, or 1.0ppm, 6h/day for two days to establish concentration-related effects on glucose tolerance and lung injury. In a second experiment, rats were exposed to FA or 1.0ppm O3, 6h/day for either one or two consecutive days, and systemic metabolic responses were determined immediately after or 18h post-exposure. O3 increased serum glucose and leptin on day 1. Glucose intolerance persisted through two days of exposure but reversed 18h-post second exposure. O3 increased circulating metabolites of glycolysis, long-chain free fatty acids, branched-chain amino acids and cholesterol, while 1,5-anhydroglucitol, bile acids and metabolites of TCA cycle were decreased, indicating impaired glycemic control, proteolysis and lipolysis. Liver gene expression increased for markers of glycolysis, TCA cycle and gluconeogenesis, and decreased for markers of steroid and fat biosynthesis. Genes involved in apoptosis and mitochondrial function were also impacted by O3. In conclusion, short-term O3 exposure induces global metabolic derangement involving glucose, lipid, and amino acid metabolism, typical of a stress-response. It remains to be examined if these alterations contribute to insulin resistance upon chronic exposure.

The cellular and genomic response of rat dopaminergic neurons (N27) to coated nanosilver.

This study examined if nanosilver (nanoAg) of different sizes and coatings were differentially toxic to oxidative stress-sensitive neurons. N27 rat dopaminergic neurons were exposed (0.5-5 ppm) to a set of nanoAg of different sizes (10nm, 75 nm) and coatings (PVP, citrate) and their physicochemical, cellular and genomic response measured. Both coatings retained their manufactured sizes in culture media, however, the zeta potentials of both sizes of PVP-coated nanoAg were significantly less electronegative than those of their citrate-coated counterparts. Markers of oxidative stress, measured at 0.5-5 ppm exposure concentrations, indicated that caspase 3/7 activity and glutathione levels were significantly increased by both sizes of PVP-coated nanoAg and by the 75 nm citrate-coated nanoAg. Both sizes of PVP-coated nanoAg also increased intra-neuronal nitrite levels and activated ARE/NRF2, a reporter gene for the oxidative stress-protection pathway. Global gene expression on N27 neurons, exposed to 0.5 ppm for 8h, indicated a dominant effect by PVP-coated nanoAg over citrate. The 75 nm PVP-coated material altered 196 genes that were loosely associated with mitochondrial dysfunction. In contrast, the 10nm PVP-coated nanoAg altered 82 genes that were strongly associated with NRF2 oxidative stress pathways. Less that 20% of the affected genes were shared by both sizes of PVP-coated nanoAg. These cellular and genomic findings suggest that PVP-coated nanoAg is more bioactive than citrate-coated nanoAg. Although both sizes of PVP-coated nanoAg altered the genomic expression of N27 neurons along oxidative stress pathways, exposure to the 75 nm nanoAg favored pathways associated with mitochondrial dysfunction, whereas the 10nm PVP-coated nanoAg affected NRF2 neuronal protective pathways.

Meta-analysis of gene expression in the mouse liver reveals biomarkers associated with inflammation increased early during aging.

Aging is associated with a loss of cellular homeostasis, a decline in physiological function and an increase in various pathologies. Employing a meta-analysis, hepatic gene expression profiles from four independent mouse aging studies were interrogated. There was little overlap in the number of genes or canonical pathways perturbed, suggesting that independent study-specific factors may play a significant role in determining age-dependent gene expression. However, 43 genes were consistently altered during aging in three or four of these studies, including those that (1) exhibited progressively increased expression starting from 12 months of age, (2) exhibited similar expression changes in models of progeria at young ages and dampened or no changes in old longevity mouse models, (3) were associated with inflammatory tertiary lymphoid neogenesis (TLN) associated with formation of ectopic lymphoid structures observed in chronically inflamed tissues, and (4) overlapped with genes perturbed by aging in brain, muscle, and lung. Surprisingly, around half of the genes altered by aging in wild-type mice exhibited similar expression changes in adult long-lived mice compared to wild-type controls, including those associated with intermediary metabolism and feminization of the male-dependent gene expression pattern. Genes unique to aging in wild-type mice included those linked to TLN.

Transcriptional ontogeny of the developing liver.

BACKGROUND: During embryogenesis the liver is derived from endodermal cells lining the digestive tract. These endodermal progenitor cells contribute to forming the parenchyma of a number of organs including the liver and pancreas. Early in organogenesis the fetal liver is populated by hematopoietic stem cells, the source for a number of blood cells including nucleated erythrocytes. A comprehensive analysis of the transcriptional changes that occur during the early stages of development to adulthood in the liver was carried out. RESULTS: We characterized gene expression changes in the developing mouse liver at gestational days (GD) 11.5, 12.5, 13.5, 14.5, 16.5, and 19 and in the neonate (postnatal day (PND) 7 and 32) compared to that in the adult liver (PND67) using full-genome microarrays. The fetal liver, and to a lesser extent the neonatal liver, exhibited dramatic differences in gene expression compared to adults. Canonical pathway analysis of the fetal liver signature demonstrated increases in functions important in cell replication and DNA fidelity whereas most metabolic pathways of intermediary metabolism were under expressed. Comparison of the dataset to a number of previously published microarray datasets revealed 1) a striking similarity between the fetal liver and that of the pancreas in both mice and humans, 2) a nucleated erythrocyte signature in the fetus and 3) under expression of most xenobiotic metabolism genes throughout development, with the exception of a number of transporters associated with either hematopoietic cells or cell proliferation in hepatocytes. CONCLUSIONS: Overall, these findings reveal the complexity of gene expression changes during liver development and maturation, and provide a foundation to predict responses to chemical and drug exposure as a function of early life-stages.

Hepatic xenobiotic metabolizing enzyme and transporter gene expression through the life stages of the mouse.

BACKGROUND: Differences in responses to environmental chemicals and drugs between life stages are likely due in part to differences in the expression of xenobiotic metabolizing enzymes and transporters (XMETs). No comprehensive analysis of the mRNA expression of XMETs has been carried out through life stages in any species. RESULTS: Using full-genome arrays, the mRNA expression of all XMETs and their regulatory proteins was examined during fetal (gestation day (GD) 19), neonatal (postnatal day (PND) 7), prepubescent (PND32), middle age (12 months), and old age (18 and 24 months) in the C57BL/6J (C57) mouse liver and compared to adults. Fetal and neonatal life stages exhibited dramatic differences in XMET mRNA expression compared to the relatively minor effects of old age. The total number of XMET probe sets that differed from adults was 636, 500, 84, 5, 43, and 102 for GD19, PND7, PND32, 12 months, 18 months and 24 months, respectively. At all life stages except PND32, under-expressed genes outnumbered over-expressed genes. The altered XMETs included those in all of the major metabolic and transport phases including introduction of reactive or polar groups (Phase I), conjugation (Phase II) and excretion (Phase III). In the fetus and neonate, parallel increases in expression were noted in the dioxin receptor, Nrf2 components and their regulated genes while nuclear receptors and regulated genes were generally down-regulated. Suppression of male-specific XMETs was observed at early (GD19, PND7) and to a lesser extent, later life stages (18 and 24 months). A number of female-specific XMETs exhibited a spike in expression centered at PND7. CONCLUSIONS: The analysis revealed dramatic differences in the expression of the XMETs, especially in the fetus and neonate that are partially dependent on gender-dependent factors. XMET expression can be used to predict life stage-specific responses to environmental chemicals and drugs.

Biomarkers of acute respiratory allergen exposure: screening for sensitization potential.

Effective hazard screening will require the development of high-throughput or in vitro assays for the identification of potential sensitizers. The goal of this preliminary study was to identify potential biomarkers that differentiate the response to allergens vs non-allergens following an acute exposure in naïve individuals. Female BALB/c mice received a single intratracheal aspiration exposure to Metarhizium anisopliae crude antigen (MACA) or bovine serum albumin (BSA) in Hank's Balanced Salt Solution (HBSS) or HBSS alone. Mice were terminated after 1, 3, 6, 12, 18 and 24 h. Bronchoalveolar lavage fluid (BALF) was evaluated to determine total and differential cellularity, total protein concentration and LDH activity. RNA was isolated from lung tissue for microarray analysis and qRT-PCR. MACA administration induced a rapid increase in BALF neutrophils, lymphocytes, eosinophils and total protein compared to BSA or HBSS. Microarray analysis demonstrated differential expression of genes involved in cytokine production, signaling, inflammatory cell recruitment, adhesion and activation in 3 and 12 h MACA-treated samples compared to BSA or HBSS. Further analyses allowed identification of approximately 100 candidate biomarker genes. Eleven genes were selected for further assessment by qRT-PCR. Of these, 6 demonstrated persistently increased expression (Ccl17, Ccl22, Ccl7, Cxcl10, Cxcl2, Saa1), while C3ar1 increased from 6-24 h. In conclusion, a single respiratory exposure of mice to an allergenic mold extract induces an inflammatory response which is distinct in phenotype and gene transcription from the response to a control protein. Further validation of these biomarkers with additional allergens and irritants is needed. These biomarkers may facilitate improvements in screening methods.

Analysis of the heat shock response in mouse liver reveals transcriptional dependence on the nuclear receptor peroxisome proliferator-activated receptor alpha (PPARalpha).

BACKGROUND: The nuclear receptor peroxisome proliferator-activated receptor alpha (PPARalpha) regulates responses to chemical or physical stress in part by altering expression of genes involved in proteome maintenance. Many of these genes are also transcriptionally regulated by heat shock (HS) through activation by HS factor-1 (HSF1). We hypothesized that there are interactions on a genetic level between PPARalpha and the HS response mediated by HSF1. RESULTS: Wild-type and PPARalpha-null mice were exposed to HS, the PPARalpha agonist WY-14,643 (WY), or both; gene and protein expression was examined in the livers of the mice 4 or 24 hrs after HS. Gene expression profiling identified a number of Hsp family members that were altered similarly in both mouse strains. However, most of the targets of HS did not overlap between strains. A subset of genes was shown by microarray and RT-PCR to be regulated by HS in a PPARalpha-dependent manner. HS also down-regulated a large set of mitochondrial genes specifically in PPARalpha-null mice that are known targets of PPARgamma co-activator-1 (PGC-1) family members. Pretreatment of PPARalpha-null mice with WY increased expression of PGC-1beta and target genes and prevented the down-regulation of the mitochondrial genes by HS. A comparison of HS genes regulated in our dataset with those identified in wild-type and HSF1-null mouse embryonic fibroblasts indicated that although many HS genes are regulated independently of both PPARalpha and HSF1, a number require both factors for HS responsiveness. CONCLUSIONS: These findings demonstrate that the PPARalpha genotype has a dramatic effect on the transcriptional targets of HS and support an expanded role for PPARalpha in the regulation of proteome maintenance genes after exposure to diverse forms of environmental stress including HS.

Regulation of Proteome Maintenance Gene Expression by Activators of Peroxisome Proliferator-Activated Receptor α.

The nuclear receptor peroxisome proliferator-activated receptor α (PPARα) is activated by a large number of xenobiotic and hypolipidemic compounds called peroxisome proliferator chemicals (PPCs). One agonist of PPARα (WY-14,643) regulates responses in the mouse liver to chemical stress in part by altering expression of genes involved in proteome maintenance (PM) including protein chaperones in the heat shock protein (Hsp) family and proteasomal genes (Psm) involved in proteolysis. We hypothesized that other PPARα activators including diverse hypolipidemic and xenobiotic compounds also regulate PM genes in the rat and mouse liver. We examined the expression of PM genes in rat and mouse liver after exposure to 7 different PPCs (WY-14,643, clofibrate, fenofibrate, valproic acid, di-(2-ethylhexyl) phthalate, perfluorooctanoic acid, and perfluorooctane sulfonate) using Affymetrix microarrays. In rats and mice, 174 or 380 PM genes, respectively, were regulated by at least one PPC. The transcriptional changes were, for the most part, dependent on PPARα, as most changes were not observed in similarly treated PPARα-null mice and the changes were not consistently observed in rats treated with activators of the nuclear receptors CAR or PXR. In rats and mice, PM gene expression exhibited differences compared to typical direct targets of PPARα (e.g., Cyp4a family members). PM gene expression was usually delayed and in some cases, it was transient. Dose-response characterization of protein expression showed that Hsp86 and Hsp110 proteins were induced only at higher doses. These studies demonstrate that PPARα, activated by diverse PPC, regulates the expression of a large number of genes involved in protein folding and degradation and support an expanded role for PPARα in the regulation of genes that protect the proteome.

Characterization of peroxisome proliferator-activated receptor alpha--independent effects of PPARalpha activators in the rodent liver: di-(2-ethylhexyl) phthalate also activates the constitutive-activated receptor.

Peroxisome proliferator chemicals (PPC) are thought to mediate their effects in rodents on hepatocyte growth and liver cancer through the nuclear receptor peroxisome proliferator-activated receptor (PPAR) alpha. Recent studies indicate that the plasticizer di-(2-ethylhexyl) phthalate (DEHP) increased the incidence of liver tumors in PPARalpha-null mice. We hypothesized that some PPC, including DEHP, induce transcriptional changes independent of PPARalpha but dependent on other nuclear receptors, including the constitutive-activated receptor (CAR) that mediates phenobarbital (PB) effects on hepatocyte growth and liver tumor induction. To determine the potential role of CAR in mediating effects of PPC, a meta-analysis was performed on transcript profiles from published studies in which rats and mice were exposed to PPC and compared the profiles to those produced by exposure to PB. Valproic acid, clofibrate, and DEHP in rat liver and DEHP in mouse liver induced genes, including Cyp2b family members that are known to be regulated by CAR. Examination of transcript changes by Affymetrix ST 1.0 arrays and reverse transcription-PCR in the livers of DEHP-treated wild-type, PPARalpha-null, and CAR-null mice demonstrated that (1) most (approximately 94%) of the transcriptional changes induced by DEHP were PPARalpha-dependent, (2) many PPARalpha-independent genes overlapped with those regulated by PB, (3) induction of genes Cyp2b10, Cyp3a11, and metallothionine-1 by DEHP was CAR dependent but PPARalpha-independent, and (4) induction of a number of genes (Cyp8b1, Gstm4, and Gstm7) was independent of both CAR and PPARalpha. Our results indicate that exposure to PPARalpha activators including DEHP leads to activation of multiple nuclear receptors in the rodent liver.