Temporal changes in gene expression in the liver of male plaice (Pleuronectes platessa) in response to exposure to ethynyl oestradiol analysed by macroarray and Real-Time PCR

Suppression subtractive hybridisation (SSH) was used to generate cDNA libraries representing genes differentially-expressed in liver from male plaice (Pleuronectes platessa) exposed to ethynyl oestradiol (EE2). BLAST analysis and alignments of the clones with database sequence suggested at least three vitellogenin (VTG) genes and three zona radiata protein (ZRP) genes were represented. Clones with unique sequence (62 up-, 13 down-regulated) were arrayed as probes on nylon membranes to investigate temporal expression of oestrogen-responsive genes in experimental animals. Arrays were hybridised with radiolabelled cDNAs prepared from hepatic mRNA from animals treated with EE2 for various times upto 21 days and from treated animals transferred to clean water for upto a further 31 days. By day 21 of treatment 11 out of 17 probes from unidentified genes, 21/22 VTG, 13/14 ZRP, 2/2 liver aspartic proteinase (LAP) and 8/10 other gene sequences were induced by EE2 exposure. Of the down-regulated sequences, only three showed significant, decreased expression and these encode cytochrome b and two with cryptic functions. Based on the pattern of temporal response the up-regulated probes fell into two classes. Pattern A reached maximum expression by day 16 of exposure and then declined prior to removal of EE2 at 21 days. Pattern B genes reached maximal expression between day 16 and 22, declining only after removal of EE2. Independent investigation of the expression patterns of selected probes using quantitative Real-Time PCR reproduced the distinctive patterns. The results indicate a previously unrecognised mechanism for oestrogenic toxicity in which there is a selective down-regulation of some egg proteins, potentially diminishing the quality of eggs and this may contribute to reproductive failure described elsewhere.     

Organization and repression by juvenile hormone of a vitellogenin gene cluster in the crustacean, Daphnia magna

Two Daphnia magna vitellogenin (VTG) genes in neighboring but opposite orientations were identified. One was the gene for DmagVTG1, a previously characterized VTG polypeptide with a superoxide dismutase (SOD)-like domain at its NH(2)-terminus [Kato et al., Gene 334 (2004) 157-165]. Both genes had a 17-exon and 16-intron structure in the same configuration. DmagVTG2, a polypeptide encoded by the other gene, also had a SOD-like domain at its NH(2)-terminus. The amino acid sequences of the two VTG domains were highly homologous (95.5% identity), while those of the SOD-like domains were less homologous (62.4% identity). The VTG domains are phylogenetically related to insect VTGs while the SOD-like domains are related to viral and bacterial SODs. The intergenic region of 2.6kb between the two genes contains sequences resembling known juvenile hormone (JH)-responsive and ecdysone-responsive elements. JH agonists, pyriproxyfen and fenoxycarb, strongly repressed the expression of VTG genes in neonate daphnids.     

Reciprocal inhibiting interactive mechanism between the estrogen receptor and aryl hydrocarbon receptor signaling pathways in goldfish (Carassius auratus) exposed to 17β-estradiol and benzo[a]pyrene

In the aquatic environment, both the estrogen receptor (ER) and aryl hydrocarbon receptor (AhR) responses are established biomarkers for assessing exposure to pollutants. These receptor responses can also be affected by the presence of other classes of pollutants and may result in misinterpretation of existing pollution. In this study, we investigated the interaction between ER-vitellogenin (VTG) and AhR-cytochrome P450 1A (CYP1A) signaling pathways in goldfish (Carassius auratus) after 10 days exposure to pollutants. 17β-Estradiol (E(2)) and benzo[a]pyrene (BaP) were selected as the ER and AhR agonists, respectively. The messenger RNA (mRNA) expression of ER-VTG and AhR-CYP1A in liver was determined using quantitative real-time polymerase chain reaction (QRT-PCR). VTG, endogenous E(2) and 7-ethoxyresorufin-O-deethylase (EROD) were also studied. Exposure to E(2) and BaP alone significantly induced the gene expression of ERα-VTG and AhR2-CYP1A, respectively. Moreover, the obvious expression of related proteins was also observed. However, these inductions were significantly reduced after combined exposure to E(2) and lower concentrations of BaP (20 and 50 μg/L), indicative of a reciprocal inhibiting ER-AhR interaction. However, high concentrations (100 μg/L) of BaP did not affect the E(2)-induced gene expression. Changes in VTG protein were in accordance with the expression of VTG mRNA, and more VTG protein was observed in liver than in serum. The induced endogenous E(2) levels were suppressed by the presence of BaP. While the gene expression of CYP1A showed a concentration-dependent increase, EROD induction exhibited a bell-shaped concentration-response curve. Taken together, these results demonstrate a reciprocal inhibiting mode of ER-AhR interactions and may lead to a possible underestimation of actual exposure.     

Fundamentals of DNA hybridization arrays for gene expression analysis

DNA hybridization arrays [also known as macroarrays, microarrays and/or high-density oligonucleotide arrays (Gene Chips)] bring gene expression analysis to a genomic scale by permitting investigators to simultaneously examine changes in the expression of literally thousands of genes. For hybridization arrays, the general approach is to immobilize gene-specific sequences (probes) on a solid state matrix (nylon membranes, glass microscope slides, silicon/ceramic chips). These sequences are then queried with labeled copies of nucleic acids from biological samples (targets). The underlying theory is that the greater the expression of a gene, the greater the amount of labeled target, and hence, the greater output signal. In spite of the simplicity of the experimental design, there are at least four different platforms and several different approaches to processing and labeling the biological samples. Moreover, investigators must also determine whether they will utilize commercially available arrays or generate their own. This review will cover the status of the hybridization array field with an eye toward underlying principles and available technologies. Future developments and technological trends will also be evaluated.     

Integrated temporal regulation of the photorespiratory pathway. Circadian regulation of two Arabidopsis genes encoding serine hydroxymethyltransferase

The photorespiratory pathway is comprised of enzymes localized within three distinct cellular compartments: chloroplasts, peroxisomes, and mitochondria. Photorespiratory enzymes are encoded by nuclear genes, translated in the cytosol, and targeted into these distinct subcellular compartments. One likely means by which to regulate the expression of the genes encoding photorespiratory enzymes is coordinated temporal control. We have previously shown in Arabidopsis that a circadian clock regulates the expression of the nuclear genes encoding both chloroplastic (Rubisco small subunit and Rubisco activase) and peroxisomal (catalase) components of the photorespiratory pathway. To determine whether a circadian clock also regulates the expression of genes encoding mitochondrial components of the photorespiratory pathway, we characterized a family of Arabidopsis serine hydroxymethyltransferase (SHM) genes. We examined mRNA accumulation for two of these family members, including one probable photorespiratory gene (SHM1) and a second gene expressed maximally in roots (SHM4), and show that both exhibit circadian oscillations in mRNA abundance that are in phase with those described for other photorespiratory genes. In addition, we show that SHM1 mRNA accumulates in light-grown seedlings, although this response is probably an indirect consequence of the induction of photosynthesis and photorespiration by illumination.     

Toxicogenomics of resveratrol in rat liver

Resveratrol, a polyphenolic compound found in grape skin and peanuts has been shown to prevent many diseases including cardiovascular diseases and cancer. To better understand resveratrol's potential in vivo toxicity, we studied the dose response using cDNA stress arrays coupled with drug metabolizing enzymatic (DME) assays to investigate the expression of stress-responsive genes and Phase I and II detoxifying enzymes in rat livers. Male and female CD rats were treated with high doses of resveratrol (0.3, 1.0 and 3.0 gm/kg/day) for a period of 28 days. Total RNA from rat liver was reverse-transcribed using gene-specific primers and hybridized to stress-related cDNA arrays. Among female rats, Phase I DME genes were repressed at 0.3 and 1.0 gm/kg/day doses, while genes such as manganese superoxide dismutase, cytochrome P450 reductase, quinone oxidoreductase and thiosulfate sulfurtransferase demonstrated a dose-dependent increase in gene expression. The modulation of these liver genes may implicate the potential toxicity as observed among the rats at the highest dose level of resveratrol. Real-Time PCR was conducted on some of the Phase II DME genes and anti-oxidant genes to validate the cDNA array data. The gene expression from real-time PCR demonstrated good correlation with the cDNA array data. UGT1A genes were amongst the most robustly induced especially at the high doses of resveratrol. We next performed Phase I and Phase II enzymatic assays on cytochrome P450 2E1 (CYP2E1), cytochrome P450 1A1 (CYP1A1), NAD(P)H:quinone oxidoreductase (NQO1), glutathione S-transferase (GST) and UDP-glucuronosyl transferase (UGT). Induction of Phase II detoxifying enzymes was most pronounced at the highest dose of resveratrol. CYP1A1 activity demonstrated a decreasing trend among the 3 dose groups and CYP2E1 activity increased marginally among female rats over controls. In summary, at lower doses of resveratrol there are few significant changes in gene expression whereas the modulation of liver genes at the high dose of resveratrol may implicate the potential toxicity observed.     

Endocrine disrupters with (anti)estrogenic and (anti)androgenic modes of action affecting reproductive biology of Xenopus laevis: I. Effects on sex steroid levels and biomarker expression

Adult Xenopus laevis were exposed in vivo to ethinylestradiol, tamoxifen, methyldihydrotestosterone and flutamide as (anti)estrogenic and (anti)androgenic compounds, respectively, for four weeks at a concentration of 10(-8) M and to Lambro river water, a polluted river from Italy. Effects of the treatments were analysed by mRNA expression of retinol-binding protein (RBP), transferrin (TF), transthyretin (TTR) and vitellogenin (VTG) in the liver of male and female X. laevis, to analyse the potential of these genes to detect endocrine disrupting compounds (EDC) with different modes of action. In addition, plasma VTG and sex steroid levels, estradiol-17beta (E(2)) and testosterone (T), were analysed. Sex steroids were depressed by ethinylestradiol in both sexes whereas tamoxifen increased E(2) in females. The induction of VTG protein plasma levels was more pronounced at the protein level compared to hepatic VTG mRNA expression in response to estrogenic treatment but VTG mRNA expression detected both, estrogenic and antiestrogenic EDC. The mRNA expression of TF was decreased by estrogenic and increased by antiestrogenic treatment while TTR mRNA expression was down-regulated and RBP mRNA up-regulated by estrogenic exposure. The other treatments did not affect the mRNA expression of the examined genes.     

双酚A诱导雄性中国林蛙肝细胞雌激素受体表达和卵黄蛋白原合成

为探讨双酚A(bisphenol A,BPA)对雄性两栖动物肝细胞中雌激素受体(estrogenreceptor,ER)表达和卵黄蛋白原(vitellogenin,VTG)合成的影响,将中国林蛙(Ranachensinensis)雄性成体分别持续暴露于10-7、10-6、10-5mol/LBPA水体中10、20、30d,设10-9、10-8mol/L雌二醇(E2)为阳性对照。用原位杂交技术检测ERmRNA在肝细胞中的表达定位,用免疫组织化学技术检测肝细胞中ER和VTG蛋白的表达。结果显示,各BPA和E2处理组肝细胞中均有ERmRNA阳性反应,ER和VTG蛋白的表达相对值均显著高于空白对照组。在同一暴露时间,ER和VTG表达值是随着BPA浓度的增加呈增高趋势。在同一BPA浓度,VTG表达值是随暴露时间的延长呈增高趋势,而ER表达值则无显著性变化。这些表明BPA可以通过诱导雄性中国林蛙肝细胞的ER高调导致VTG合成,但其效应远低于E2。     

Gastric cancer cell lines AGS before and after CD40 signal activating

The aim of this study was to investigate the molecular mechanisms underlying the antitumour effects of CD40L through analysing the change of genes expression profile in AGS using Affymetrix Gene Chip. Human gastric carcinoma AGS cells were first incubated with 2 μg/ml sCD40L or equal volume of medium (control) in F12 medium. RNA was isolated from AGS and were reverse transcribed, labeled with digoxigenin-11-dUTP, and then hybridized with Clontech Atlas mouse cDNA expression arrays for comparison. Performing clustering analysis, we found that 7 detected genes were down-regulated and 38 were upregulated as the sCD40L acted on AGS. To further verify the results of gene chip screening, Gene Database was searched, finding that the most significantly up-regulated genes were Gadd45a, c-Jun and Bcl-2, and the most significantly down-regulated genes were Cyclin D1, CDC6, TNFR10B, c-IAP2 and ORC5L. Based upon these findings, the signalling pathways that possibly mediate CD40-induced apoptosis are proposed.     

Gene selection in microarray data: the elephant,the blind men and our algorithms

Gene expression array data provide shadows of intricate cellular processes. Learning how to make the most of the information present in expression arrays has become a discipline in itself. In recent years, there has been an explosion of methods that analyze gene expression arrays to produce long lists of genes that express differentially in distinct cellular states. These lists will have to be organized, and the algorithms that produced them combined, if we wish to piece together the rich cellular structures probed by this high-throughput technology. Researchers will have to understand the benefits and limitations of the many existing methods to produce the combination of algorithms that best suits their gene expression experiments.     

Monitoring cellular responses to Listeria monocytogenes with oligonucleotide arrays

Listeria monocytogenes is a pathogenic intracellular microorganism whose infection induces pleiotropic biological changes associated with host cell gene expression regulation. Here we define the gene expression profiles of the human promyelocytic THP1 cell line before and after L. monocytogenes infection. Gene expression was measured on a large scale via oligonucleotide microarrays with probe sets corresponding to 6,800 human genes. We assessed and discussed the reproducibility of the hybridization signatures. In addition to oligonucleotide arrays, we also performed the large scale gene expression measurement with two high-density membranes, assaying for 588 and 18,376 human genes, respectively. This work allowed the reproducible identification of 74 up-regulated RNAs and 23 down-regulated RNAs as a consequence of L. monocytogenes infection of THP1. The reliability of these data was reinforced by performing independent infections. Some of these detected RNAs were consistent with previous results, while some newly identified RNAs encode gene products that may play key roles in L. monocytogenes infection. These findings will undoubtedly enhance the understanding of L. monocytogenes molecular physiology and may help identify new therapeutic targets.     

Effects of a verbenachalcone derivative on neurite outgrowth, inhibition of caspase induction and gene expression

A verbenachalcone derivative was synthesized and shown to protect N2a cells from caspase induction caused by serum starvation and to enhance the effect of NGF on neurite outgrowth in PC12 cells. As an initial investigation of the compound's mechanism(s) of action, we performed differential gene expression profiling in PC12 cells using oligonucleotide ( approximately 10,000 gene probes) microarrays. Gene expression patterns were compared in the presence of NGF (2 and 50 ng/mL) and NGF (2 ng/mL) plus the verbenachalcone derivative. Ten genes were significantly (2-fold; p0.05) up-regulated and seven genes were significantly down-regulated in the presence of the compound. These results were independently validated by quantitative real-time PCR for a subset of genes (cathepsin L, sigma-1 receptor and protein tyrosine phosphatase receptor type R). These genes or their protein products may represent useful therapeutic targets for treating neurodegeneration, such as Alzheimer's disease.     

Sandwich ELISAs for quantification of Xenopus laevis vitellogenin and albumin and their application to measurement of estradiol-17 beta effects on whole animals and primary-cultured hepatocytes

Sandwich enzyme-linked immunosorbent assay (ELISA) was developed for quantification of vitellogenin (VTG) and albumin (ALB) in Xenopus laevis. Working ranges of the ELISAs were 2-1000 ng/ml for VTG and 1-300 ng/ml for ALB. Recoveries of plasma VTG by ELISA were over 90% in dilutions of more than 200 times. The VTG-inducing activity of estradiol-17beta (E2) was measured in whole animals and primary cultured hepatocytes. Immersion of mature male animals in more than 1 nM E2 induced a detectable amount of plasma VTG. VTG induction in younger animals was less potent than in the mature animals but the youngest animals (1.5-3 g body mass) was applicable to the exposure test, irrespective of sex. In vitro exposure of hepatocytes to more than 0.1 nM E2 dose-dependently induced secretion of VTG into the culture medium, while ALB secretion was not significantly affected by E2 treatment. When the VTG-induction levels were normalized by use of a concentration ratio of VTG to ALB, the values obtained from three independent experiments were mutually comparable irrespective of differences in cell density and hepatocyte preparation. Thus, this ratio is thought to be useful for large-scale in vitro screening of estrogenic activities of chemical substances.     

Gene expression changes associated with cytotoxicity identified using cDNA arrays

In order to investigate gene expression changes associated with cytotoxicity, we used cDNA arrays to monitor the expression of over 5,000 genes in response to toxic stress in the HepG2 liver cell line. Cells were treated with cytotoxic doses of acetaminophen, caffeine or thioacetamide for nine time points ranging from 1 to 24 h. Samples of mRNA from each time point were used to prepare radiolabeled cDNA, which was hybridized to nylon-membrane-based cDNA arrays. High-stringency washes were applied to reduce cross-hybridization. Analysis of spot intensities revealed that each compound led to approximately 150-250 gene expression changes that were sustained over at least three adjacent time points. The affected genes could be classified into clusters based on their temporal patterns of differential expression. A common set of 44 genes showed similar expression changes in response to all three compounds. Of these changes, 90% could be confirmed by quantitative RT-PCR analysis. The results indicate that detailed array-based time-course studies, coupled with a sensitive and highly specific confirmation assay, provide a powerful means of identifying cytotoxicity-associated gene expression changes. Electronic Publication