P062 (AAA-1117-816195) Development and Characterization of Japanese Medaka (Oryzias Latipes) Random cDNA microarray for Toxicogenomic Analysis.
Start time: 8:00 AM
Gu, M.B.², Park, K.S.¹, Kim, H.¹, Youn, C.H.¹, ² Graduate School of Biotechnology, Korea University, Seoul, Korea¹ National Research Laboratory on Environmental Biotechnology, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea
In our previous studies, a stress-specific functional cDNA microarray for Japanese Medaka fish had been developed by using 25 stress-related known genes, and then it had been extended for 120 stress-specific known genes. Those cDNA microarrays were used to characterize various toxic chemical impacts on fish in terms of expression profiling and trends of specific gene expression with aid of real time PCR technique. However, there have been some limit in understanding the behaviour of unknown genes and finding a new biomarkers using those functional cDNA microarrays. Therefore, in this study, random cDNA microarrays for Japanese medaka fish were developed for toxicogenomic analysis of the impacts of various chemicals or contaminants including endocrine disrupting chemicals, and other toxic chemicals. In the construction of random cDNA microarray, complete sequence information and annotation of genes are not required but may allow to have many spots of cDNAs. In this study, about 2000 random cDNA library genes were spotted on the chip. In this presentation, the construction and characterization of this chip for model chemicals will be presented.

P063 (AAA-1117-819337) Gene Expression in Medaka Embryo and Fry Exposed to EDCs and New program for Monitoring.
Start time: 8:00 AM
HU, JY¹, Zhang, ZB¹, Sai, LL¹, Hou, YF¹, ¹ College of Environmental Science, Peking University, Beijing, Beijing, China
A number of synthetic as well as natural chemicals are capable of interfering with sex differentiation and development of wildlife, including fish, amphibians, reptiles, birds, and mammals. These so-called endocrine disrupting chemicals (EDCs) may lead to sex retarding, intersex or sex reverse. Many studies have demonstrated that the early development (including embryo and larva) of animal are the most susceptible stage to EDCs exposure. So we established the method of isochronous isolation of total RNA and genomic DNA from single embryo or fry of medaka ( Oryzias latipes ), developed method of sex indentification by genomic DNA, and investigated the differential gene expression in male or female embryo and fry exposed to different EDCs by quantitative real-time RT-PCR. And several sex differentiation related gene expressions are significantly disrupted was found. On the other hand, the in vivo experiment with embryo or fry is less expensive and with less volume than experiment with adult fish. So we will bring forward a new program for monitoring EDCs in this report.

P064 (AAA-1117-817626) Toxicogenomic Analysis of EDCs (Endocrine Disrupting chemicals) Using Japanese Medaka (Oryzias Latipes) Random cDNA Microarray.
Start time: 8:00 AM
Gu, M.B.², Park, K.S.¹, Kim, H.¹, ² Graduate School of Biotechnology, Korea University, Seoul, Korea¹ National Research Laboratory on Environmental Biotechnology, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea
In order to conduct toxicogenomic analysis of Japanese Medaka (Oryzias Latipes) fish for 17-beta estradiol (E2), Nonylphenol (NP) and Phenol (P), newly fabricated random cDNA microarray containing more than 2000 random cDNAs were used. All microarray experiments were conducted with male fish. Two different concentrations of each chemical, which were derived from acute toxicity test, were exposed to fish, and liver samples obtained at different time were used for DNA microarray experiments. The results were repeatedly confirmed for biomarker genes previously known and additional expression profiling and bioinformatic analysis have revealed new findings. The results from time course expression analysis as well as self-organizing mapping (SOM) studies will be presented with biomarker analysis.

P066 (STA-1117-848996) Microarray Analysis of Liver from Rainbow Trout (Oncorhynchus mykiss) Fed Polycyclic Aromatic Hydrocarbons.
Start time: 8:00 AM
Stanley, K¹, Bravo, C¹, Curtis, L¹, Bayne, C², Gerwick, L², Lambertini, E³, Loge, F³, Hahn, M⁴, Williams, D¹, ¹ Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, Oregon, USA² Department of Zoology, Oregon State University, Corvallis, Oregon, USA³ Department of Civil and Environmental Engineering, University of California Davis, Davis, California, USA⁴ Department of Biology, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts, USA
Polycyclic aromatic hydrocarbons (PAHs) are present in aquatic ecosystems where they commonly exist as complex mixtures. Exposure to individual PAHs is known to cause cancer and immunotoxicity in aquatic organisms. Laboratory experiments using environmentally relevant concentrations of PAHs are necessary to gain insight into mechanisms of toxicity in fish in the field. Aryl hydrocarbon receptor activation is one possible mechanism of PAH toxicity in fish. Rainbow trout were fed 160ppm of benzo[a]pyrene, 160ppm benzo[e]pyrene, or a 400ppm mixture of ten high molecular weight PAHs for 50 days. A vehicle control group was also included. Blood, liver and kidney were collected from fish in all treatment groups after 3, 7, 14, 28, and 50 days of feeding. DNA damage, cytochrome P450 activity and lipid peroxidation were measured using the comet assay, EROD, and isoprostanes, respectively. Percent DNA damage increased after 14 days and then decreased from this maximum after 28 or 50 days in all treatments. The same results were obtained for the cytochrome P450 measurements. Lipid peroxidation was significantly higher in the 400ppm treatment on day 50 compared to the control. The aryl hydrocarbon binding assay was conducted by incubating rainbow trout AhR2a with a 2nm solution of [3H]TCDD and 1, 10, or 100nm solutions of DMSO, benzo[a]pyrene or benzo[e]pyrene. As expected, benzo[a]pyrene competed with TCDD for binding to the receptor. Benzo[e]pyrene did not compete for binding, proposing an oxidative stress mechanism independent of AhR. Microarrays selective for rainbow trout genes involved in immune, toxicological, endocrine, and stress responses were hybridized with hepatic total RNA. Relationships between phenotypic features and PAH-dependent changes in gene expression are of special interest.

P067 (TSY-1117-852590) Radiation-induced DNA stand-breakage in Japanese medaka (Oryzias latipes).
Start time: 8:00 AM
Tsyusko, O.¹, Glenn, T.¹, Hinton, T.¹, Coughlin, D.¹, ¹ Savannah River Ecology Laboratory, The university of Georgia, Aiken, SC, USA
During the last decades, there has been a need for rapid and simple tests to evaluate the effects of exposure to environmental contaminants which can have an adverse health impacts due to DNA damage. Comet assay, a sensitive method for detecting DNA damage in individual cells, allows the detection of diverse DNA alterations including double and single strand breaks, alkali-labile sites, and incomplete repair sites. By quantifying strand breaks for relatively many cells, standard statistical approaches can be used to quantify differences within and among individuals and treatments. This method was used to detect total DNA damage due to radiation exposure in Japanese medaka (Oryzias latipes), one of the most widely used fish in comparative mutagenesis, carcinogenesis, and genomic instability studies. The adult fish was irradiated at three different dose rates, and their red blood cells were subjected to the Comet assay. Our preliminary data showed elevated level of DNA damage in irradiated medaka cells when compared to that of control fish. Relatively minor DNA damage was observed at low-dose rates suggesting that exposure to environmentally relevant radiation doses has the potential to induce DNA strand breakage in medaka. The amount of the DNA damage increased with increasing dose rate. Further work is necessary to determine if the Comet assay can be used as bio-indicator for radiation-induced DNA damage in medaka.

P068 (VAN-1117-824415) Single and combined effects of organophosphate pesticides and nickel through microarray analysis in Daphnia magna.
Start time: 8:00 AM
Vandenbrouck, T¹, Soetaert, A¹, van der Ven, K¹, Moens, L¹, Naudts, B², Blust, R¹, De Coen, W¹, ¹ Laboratory for Ecophysiology, Biochemistry and Toxicology, Department of Biology, University of Antwerp, Antwerp, Belgium² Intelligent System Lab, Department of Mathematics and Informatics, University of Antwerp, Antwerp, Belgium
Daphnia magna is frequently used as a standard organism in laboratory toxicity testing in order to assess the potential hazards of chemicals to the aquatic environment. In this context, it is often assumed that laboratory populations of organisms will respond identically towards a certain contaminant as field populations. However, other factors such as acclimation, adaptation and present environmental conditions may produce differences in tolerance and/or sensitivity between laboratory and field-collected populations. For instance, in natural communities, additional stresses such as the presence of predators may strongly interfere with responses to pollutant stress. Moreover, relevant environmental conditions are usually characterized by the presence of multiple chemical stressors. From a toxicological point of view, these interactions can highly influence the overall impact of chemical stressors. In the present study, we evaluated the impact of multiple chemicals on D. magna using microarray analysis. In addition, multiple biomarkers were measured (Acetylcholinesterase, Cellular Energy Allocation) as well as effects on growth. Two organophosphate pesticides (chlorpyrifos, diazinon) and nickel were assessed as individual compounds and as binary mixtures. Differential gene expression was assessed using a custom cDNA microarray containing genes related to energy metabolism, molting and reproduction. Insights concerning the specific modes of action of the individual compounds and of the mixtures were gained. The potential of microarray analysis for the evaluation of mixture toxicity was assessed.

P069 (VAR-1117-827374) The Effect of Concentration on Gene Expression Profiling in Daphnia magna.
Start time: 8:00 AM
Varshavsky, J¹, Poynton, H¹, Chang, B¹, Holman, P¹, Loguinov, A¹, Bauer, D², Colbourne, J², Vulpe, C¹, ¹ UC Berkeley, Berkeley, CA, USA² Indiana University, Bloomington, IN, USA
Despite the threat that pollution poses to aquatic ecosystems, the present methods for identifying the chemicals responsible for toxicity in the environment are not specific or simplistic enough for routine monitoring. The emerging field of ecotoxicogenomics involves using gene expression profiling to both sensitively indicate stress to an ecosystem and identify the casual agents. We have shown that the metals copper, cadmium, and zinc cause the differential expression of unique subsets of genes and have identified gene expression profiles for these metals at the 1/10 LC50. However, different concentrations of metals present in the environment will influence the gene expression profiles for these metals. We have performed microarray hybridizations using high (acute No Observable Effect Concentration [NOEC]) and low (chronic 1/10 EC50) concentrations for copper, cadmium, and zinc. We will compare the similarities and differences in gene expression at the different concentrations. In addition, we will address the sensitivity of gene expression profiling and determine whether a microarray-based assay can detect the presence of metals at levels below those that cause chronic phenotypic responses.

P070 (CRA-1117-834750) Are all p53s created equal? Uncovering the function of soft-shell clam (Mya arenaria) p53.
Start time: 8:00 AM
Crawford, Lauren¹, Butler, Rondi¹, Van Beneden, Rebecca¹, ¹ University of Maine, School of Marine Sciences, Orono, ME, 04469
The presence of gonadal tumors in the soft-shell clam, Mya arenaria, has been observed in certain eastern Maine populations. The etiology of the tumors is unknown, but environmental factors including cell cycle disrupting contaminants are likely involved. We are particularly interested in the tumor suppressor protein p53, which functions as a transcription factor in cell cycle regulation. In response to DNA damage, stabilized p53 protein can activate pathways leading to either cell cycle arrest or apoptosis, thereby preventing the perpetuation of damaged DNA. If mutated, p53 loses its protective function, potentially leading to tumor formation. Mya arenaria p53 (Map53) has been identified through sequence similarity to known vertebrate and invertebrate p53s. Earlier histological and gene expression studies linked an undifferentiated cell state in clam gonadal tissue with altered p53 protein levels. These cells were noted in both field-collected tumor-bearing clams and clams exposed in the laboratory to 2,4-D. Map53 shows sequence homology to H. sapiens p53 (Hsp53) in key functional domains. In order to assess functional similarity, vectors having a constituitive promoter were constructed using Map53 or wild-type Hsp53 cDNA, and transfected into the p53 null cell line H1299. The expression of p53, p21^WAF1/CIP1, and actin was determined by western blot analysis. p21^WAF1/CIP1, when upregulated by p53, is known to lead to growth arrest. The ability of Map53 to induce apoptosis was evaluated by caspase-3 activation. Preliminary results show an increase in cellular p21^WAF1/CIP1 protein levels in both human and clam p53-expressing H1299 cells and no activation of caspase-3. This suggests that both human and clam p53 are functioning in a similar manner to lead the H1299 cells into growth arrest. Uncovering Map53 function will contribute to the understanding of its role in clam gonadal tumorigenesis.

P071 (RAM-1117-835185) Pendrin gene expression in the gonads of deer mice following exposure to ammonium perchlorate.
Start time: 8:00 AM
Ramachandran, B¹, Viñas, R¹, Gentles, A¹, Cox, S¹, Smith, E¹, ¹ The Institute of Environmental and Human Health, Texas Tech University, Lubbock, Texas, USA.
Pendrin is a membrane transport protein, which functions in the transport of chloride, bicarbonate, and iodide. Perchlorate, a component of fuel for rockets and missiles, is a known disruptor of thyroid hormone homeostasis and iodide transport. The objective of this study was to characterize pendrin gene expression and evaluate the effects of ammonium perchlorate (AP) on pendrin expression in the gonads of deer mice, a sentinel wildlife species. Deer mice were exposed to AP (0, 58.5 and 117 ppm) either during gestation-alone (GA) or gestation and lactation (GL). The results indicate that conception was 100% in the control group while it was 75% and 58.33% in the low and high treatment groups, respectively. Litter size was significantly lower (p<0.05) in the highest AP exposure group compared to the other groups. Interestingly, the highest exposure group had the highest pup survival percentage through postnatal day (PND) 21. At the end of exposure the pups were euthanized at PND 21 and 45 for tissue collection. A partial pendrin cDNA sequence was generated by RT-PCR. Taqman specific probe and primers were designed from this cDNA sequence for real-time PCR quantification of mRNA equivalents for pendrin gene expression. The expression profile was standardized to GAPDH. The data indicated that exposure to AP during GA and GL, significantly (p<0.05) suppressed pendrin gene expressions in the treatment groups at PND 21 and PND 45 in the ovaries. In the testes of PND 45 males following exposure to AP (58.5 ppm) during GL, pendrin gene expression was 1.5 fold higher than controls. Similar results were observed for the males of PND 45 GA exposure (117 ppm) group. The data indicates that exposure to perchlorate during GA and GL alters gene expression in the gonads of developing deer mice. This study shows that perchlorate is a potential developmental toxicant at the molecular level and raises the implication of the role of perchlorate in trans-generational effects.

P072 (LEE-1117-806460) Development of a Gene Expression Screening Assay Using Zebrafish Exposed to Model Estrogens.
Start time: 8:00 AM
Lee, D¹, Hoffmann, J¹, Brill, J¹, Price, B¹, Versteeg, D¹, ¹ The Procter and Gamble Co., Cincinnati, Ohio, USA
Many natural and man-made compounds have been shown to affect the endocrine status of fish, and a variety of approaches are being developed to assay for endocrine activity. To date, most approaches tend to be cumbersome, limited in scope or prohibitively expensive to carry out with the dozens and perhaps thousands of compounds that may need to be tested. Our goal is to develop a rapid screening assay for endocrine disrupting compounds using q-rt-PCR. Using model endocrine active compounds and the zebrafish Affymetrix GeneChip, our strategy is to identify a suite of differentially expressed genes. We report our studies with female, adult zebrafish (Danio rerio) exposed to 17 -ethinylestradiol (EE2). A variety of genes were identified that were differentially regulated in a dose and time-dependent manner. Genes selected were involved in retinoic acid metabolism, steroid hormone/action, insulin metabolism and cell cycle. Exposure to a model weak estrogen, t-pentylphenol, will be used to validate the discrimination capabilities of the selected genes. This process will be completed for other modes of endocrine activity (e.g., androgenicity, antiestrogenicity, etc.) to develop a suite of genes diagnostic of a range of endocrine modes of action. A procedure for using the gene expression screening assay using the q-rt-PCR with compounds of unknown activity will be discussed. The ultimate goal is to employ this suite of genes with a zebrafish hepatocyte immortalized cell line to develop in vitro screens for multiple chemicals mechanisms.

P073 (KIM-1117-816780) Gene expression characteristics of Vitellogenin, Choriogenin L, Aryl Hydrocarbon Receptor, Cytochrome P450 1A and Heat Shock Protein 70 in Japanese medaka fish exposed to Endocrine Disrupting Chemicals.
Start time: 8:00 AM
Kim, H.N., Park, K.S., Gu, M.B.,
In the our previous study, the gene expression kinetics of estrogen receptor(ER), cytochrome P450 aromatase(CYP19) and p53 were examined for the representative endocrine disrupting chemicals (EDCs), 17 beta estradiol, nonylphenol and bisphenol A. Each biomarker is known by its estrogenic potential, steroidgenesis of chemicals and tumor suppression, respectively. In this study, expression kinetics of the female related genes and other cellular toxicity represented genes, such as vitellogenin(yolk protein precursor), choriogenin L(inner membrane precursor of egg), aryl hydrocarbon receptor (AhR), cytochrome P450 1A (CYP1A) and heat shock protein 70(HSP70) were studied on male Japanese medaka in the presence of three EDCs. Japanese medaka was exposed to 100ppb and 1ppb of 17 beta estradiol, 75ppb and 7.5ppb of nonylphenol and 75ppb and 7.5ppb of bisphenol A. The highest concentration tested with each chemical was LC 20 value obtained from acute toxicity test on young Japanese medaka. The fish livers were extracted after 1day, 2days and 4days from exposure. Gene expressions were quantified by measurement of messenger RNA (mRNA) in liver extracts using Taqman based real time PCR method. From the expression patterns of these five genes, the different transcriptional mechanisms and the stability of the transcripts of each gene were examined for each chemical.

P074 (KIL-1117-733691) Screening of teleost cell lines for bystander effects of low–dose radiation – preliminary results of a 5-year study.
Start time: 8:00 AM
Kilemade, M.¹, O'Neill, A.¹, Mothersill, C.¹, Seymour, C.¹, ¹ McMaster University, Hamilton, Ontario, Canada
Bystander effects are detected in cells, which were not themselves exposed to radiation but were exposed to signals from exposed (irradiated) cells. They involve expression of many endpoints associated with genomic instability. Now that the existence of radiation–induced bystander effects is not in doubt, the problem for regulators is to assess their importance. Previous work by people in this group suggests that these effects dominate the low dose region of the dose–response curve, and saturate at low doses (less than 5mGy). These effects occur in many cell types and species following high–or low–linear energy transfer radiation exposures. The signals produced are capable of inducing death, genomic instability, mutations, or transformation in cells not directly hit by radiation themselves. The effect may be important in radiation protection of both human and non–human biota and may impact on low dose and low dose rate risk estimates. Bystander signals are now considered by many authors to drive genomic instability. In this study a range of teleost fish cell lines were exposed to a low radiation dose range (0.002–5 Gy). Clonogenic survival was measured in the exposed cells and in the distant progeny of exposed cells to assess early and delayed cell death. Clonogenic survival was also measured in cultured cells, which received culture medium from irradiated cells to determine bystander effects. Our preliminary data have shown that the dose response curves for the fish cells exposed to radiation demonstrated the bystander effect. These results demonstrate that damage due to low doses of radiation occurs both directly and indirectly and may have implications for radiation and environmental protection of biota.

P075 (PLA-1117-724099) Regulation of CYP enzymes and Drug Transporters by Drugs and Environmental Chemicals.
Start time: 8:00 AM
PLANT, N¹, AL-SALMAN, F², GIBSON, G³, ¹ University of Surrey, Guildford, U.K² University of Surrey, Guildford, U.K³ University of Surrey, Guildford, U.K
The body is constantly exposed to many different external chemicals (xenobiotics), be they environmental toxins or therapeutic medicines. Cytochrome P450 enzymes and drug transporters form part of the core protection system for the body, regulating entry of xenobiotics, and increasing clearance rates of chemicals that do enter the body. It is therefore important to know how these systems will respond to novel chemicals, with human cell lines being commonly used in such drug metabolism studies. In this study we have determined the transcript levels for several cytochrome P450s or drug transporter from human liver (Huh7), intestine (CaCo-2) and lung (A549) cell lines, and compared them to adult and foetal levels in the respective tissues: Basal expression of the selected CYP genes and drug transporters is significantly lower in these cell lines than in either adult or foetal primary tissue. We have also examined the effect of xenobiotic exposure on the expression of these transcripts, showing that whereas some cell lines have maintained their ability to show genome-base activations of gene expression, others are refractory , a fact that has important implications for drug studies carried out in these cell lines. Finally, we have examined how two of these genes, CYP3A4 and MDR1, are regulated in different tissues and cell types. Current evidence suggests that in certain tissues these genes show positive coordination of their response to xenobiotic exposure, whereas in other negative or no coordination is seen. Using reporter gene constructs for the proximal promoters of these two genes we are examining the molecular mechanisms underlying these complex responses to chemical exposure. Such work will help improve prediction of body responses to xenobiotic exposure, as well as increase the accuracy of current risk assessment methodologies.

P076 (LUN-1117-791080) Functional toxicogenomic approach for assessment of risks and benfits associated with a methylmercury-contaminated diet.
Start time: 8:00 AM
Lundebye, A.-¹, Glover, C.¹, Sales, G.², Xheng, D.², Hogstrand, C.², ¹ NIFES, Bergen, Norway² Kings College, London, England
Methylmercury (MeHg) contamination of seafood can have a severe impact on human helath, with neurological effects during development of particular concern. However current MeHg risk assessments are potentially flawed in that epidemiological studies often do not consider positive health effects of seafood consumption or possible amelioration of toxicity by nutritional components, while laboratory experiments often do not account for the fact that most, if not all, MeHg in the diet is found as a cysteine conjugate, which is known to be less toxic than the widely utilised MeHgCl, at least in cell culture. As part of an ongoing research programme designed to address these concerns, we have conducted a study that attempted to establish the importance of MeHg speciation, and identify the mechanisms of MeHg toxicity in a murine model. Female mice were fed MeHg (as either MeHgCl or MeHgCys; 0, 2, 5, or 10 mg/kg diet) for a total of 8 weeks (3 weeks before mating, during gestation, and for two weeks following parturition). Assessments of neurobehavioural indices such as stress/anxiety response, reflex behaviour, locomotion and ultrasonic vocalisations were made in pups born to dosed dams. Brains of these pups were then subjected to microarray analysis to monitor changes in gene expression. These measures were accompanied by examination of Hg and MeHg tissue burdens. Preliminary results from these initial experiments will be presented. This approach offers advantages in that it elucidates sensitive biomarkers of toxicity, permits simultaneos exploration of numerous biochemical pathways that may contribute towards toxicity, while linking global molecular changes directly to biochemistry, physiology, behaviour and tissue burden. This functional genomic assessment of toxicity is likely to offer both excellent potential for deciphering complex biologiocal processes while providing key information for the development of improved environmental health policy.

P077 (BUT-1117-803863) Interaction trapping of novel aryl hydrocarbon receptor (AHR) interactors from a Mya arenaria cDNA library.
Start time: 8:00 AM
Butler, R¹, Van Beneden, R¹, ¹ University of Maine, Orono, ME, USA
Increasing evidence suggests that the AHR has a role independent of dioxin binding. This is apparent given that invertebrate AHR homologues (M. arenaria, D. melanogaster and C. elegans) lack the ability to bind prototypical ligands ([³H]TCDD and [³H] -naphthoflavone). Additionally, AHR is the only member of the PAS superfamily of transcriptional regulators to be ligand activated. Taken together, one might speculate that the invertebrate AHRs may not have the same requirements as vertebrate AHRs for ligand activation. Investigation into the gonadal expression of M. arenaria AHR revealed a positive correlation between AHR protein levels and stage of ovogenesis. To better understand AHR function in the female clam reproductive tissue, a truncated AHR (amino acids 1-400) was used as the 'bait' protein to screen a yeast two-hybrid cDNA expression library prepared from the gonad of a female with mature gametes. Sequence homology analysis revealed an array of putative AHR-interacting proteins. A number of these proteins have been identified by others as essential for early zebrafish, yeast, worm and/or fly development (e.g., ribonucleoside reductase small subunit, ribosomal proteins L7 and L13, nacscent polypeptide-associated complex a subunit and zinc finger 183). Full-length sequences for proteins in this category were obtained by RACE PCR and re-evaluated by interaction trapping. Proteins found positive for interaction with AHR are currently being verified by an independent, in vitro method. We hypothesize that interaction with one or more of these critical developmental genes may indicate an endogenous function for the AHR.

P078 (SOE-1117-805257) Energy metabolism, molting and reproduction related microarray as a tool for toxicant evaluation in Daphnia magna.
Start time: 8:00 AM
Soetaert, A¹, Vandenbrouck, T¹, Moens, L¹, van der Ven, K¹, Van Leemput, K², Blust, R¹, De Coen, W¹, ¹ Laboratory for Ecophysiology, Biochemistry and Toxicology, Department of Biology, University of Antwerp, Antwerp, Belgium² Intelligent System Lab, Department of Mathematics and Informatics, University of Antwerp, Antwerp, Belgium
Since decades Daphnia magna has been the organism of choice for aquatic toxicity testing. Presently, molecular tools are emerging which enable environmental toxicologists to monitor gene expression changes resulting from toxicant exposure in animal species whose genome is not fully known, like Daphnia magna. In this study we applied a combination of genomics technologies to evaluate differential gene expression in Daphnia magna after toxicant exposure. Since chemical stress reduces the amount of energy that is allocated to growth and reproduction we have investigated the expression of genes related to energy metabolism, growth (molting) and reproduction (age-specific) for toxicant characterization. Energy metabolism, molting and reproduction specific cDNA libraries were created using the Suppression Subtractive Hybridization PCR and applied for construction of a custom cDNA array. An important fraction of the genes in these libraries are related to glycolysis, the electron transport chain, protein, carbohydrate and lipid metabolism (energy metabolism), cuticle and protein degradation (molting) and embryo development (reproduction). To evaluate this custom cDNA array exposure experiments were conducted with different chemicals (propiconazole, fenarimol, Cd and Pb) and adverse effects were studied at higher levels of biological organization. Exposure to propiconazole (a fungicide causing developmental deformities in Daphnia offspring) was used as an initial case study. Microarray analysis of freshwater fleas exposed for short periods to this compound clearly caused important effects in genes related to embryo development, energy metabolism, molting and cell cycle. One of the major affected genes was vitellogenin which plays a crucial role in the embryonic development. The downregulation of vitellogenin was also found after exposure to another embryotoxicant namely fenarimol. Organismal effects confirmed the major molecular findings. Presently, we have demonstrated the potential of microarray analysis in toxicity screening with Daphnia magna. Future applications of these molecular techniques will be discussed.

P079 (GON-1117-838737) Microarray Analysis of 2,4,6 Trinitrotoluene Exposure Effects in the Earthworm Eisenia fetida.
Start time: 8:00 AM
Guan, X¹, Gong, Ping¹, Inouye, L², Indest, Karl², Perkins, E², ¹ Analytical Services, Inc., Vicksburg, MS, USA² US Army ERDC, Vicksburg, MS, USA
Current and historical training activities of the U.S. Army have resulted in the release of munitions such as 2,4,6-trinitrotoluene (TNT) into the environment that may adversely impact soil organisms. We have developed an Eisenia fetida cDNA microarray and used it to assess the impact of TNT contaminated soil on E.fetida gene expression. The cDNA microarray was constructed from a cDNA library enriched for genes affected by cadmium, TNT, 2,6-Dinitrotoluene, HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine) and RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine). 2200 randomly picked cDNAs were spotted onto glass slides. To assess the impact of different durations of TNT exposure, time course experiments were performed at 4-d, 14-d or 28-d at 0 (control) or 100 mg/kg of TNT, a previously determined lowest observable lethal effect level for E. fetida. Three individual worm replicates were analyzed at each time point and exposure concentration in a loop design experiment. Twenty nine cDNAs were identified as significantly affected by TNT exposure. A putative mitogen-activated protein kinase involved in signal transduction was induced at 4-d. At 4-d and 14-d, exposure resulted in down regulation of genes involved in cellular protein degradation (ubiquitin activating enzyme) and oxygen transport (hemoglobin linker chain, hemoglobin subunit B2, hemoglobin chain d1) in addition to several unknown genes. After 28-d of exposure, genes are induced involved in cellular protein degradation (ubiquitin activating enzyme, lumbrokinase-3), lysosomal degradation (beta-hexosaminidase), and several unknown genes. At 28-d, down regulated genes included a putative transcription factor, a non-heme-iron oxygen transport protein, and a trichohyalin-similar gene. Lasting effects on hemoglobin genes were not seen. These data suggest that TNT inhibits oxygen transport systems at short periods of exposure, with longer exposures resulting in significant damage to cellular proteins and macromolecules.

P080 (GRO-1118-263185) Genomics and physiology of water-borne lead toxicity in freshwater organisms.
Start time: 8:00 AM
Grosell, M¹, Mager, E¹, Wintz, H², Brix, K^{1, 3}, Vulpe, C², ¹ RSMAS, University of Miami, Miami, FL, USA² UC Berkeley, USA³ EcoTox, Key Biscayne, FL, USA
Genomics and physiology of water-borne lead toxicity in freshwater organisms M. Grosell1, E. Mager1, H. Wintz2, K.V. Brix1&3, C. Vulpe2. 1 RSMAS, University of Miami, FL, USA 2 UCBerkeley,, CA, USA 3 EcoTox, Key Biscayne, FL, USA Lead (Pb) acts as a potent Ca²⁺ antagonist and water-borne Pb exposure interrupts Ca2+ homeostasis in freshwater organisms. Sublethal consequences of reduced Ca²⁺ uptake from the water include reduced growth in the pulmonate snail, Lymnaea stagnalis and disturbance of whole body Ca²⁺ balance in juvenile fathead minnow. In the pulmonate snail, inhibition of Ca²⁺ uptake and thus shell formation initiate a cascade of effects including alkalosis, and increased Cl^- uptake. Genome wide gene expression in response to sublethal Pb exposure was measured using custom made fathead minnow microarrays hybridized with labeled cDNA obtained from larval fathead minnow. Of multiple clones on the microarray that exhibited significant changes in expression in response to Pb exposure, a subset was chosen for sequencing. Of the sequenced clones, at least five genes appeared highly relevant based on observed physiological consequences of Pb exposure. Up-regulation of genes coding for Ca²⁺ binding proteins, cysteine rich proteins and super oxide dismutase were observed consistent with expectations. In addition, up-regulation of an anion exchanger III like protein was observed in Pb exposed fish. The later observation is consistent with the alkalosis and increased Cl^- uptake seen in Pb exposed snails which perhaps suggest that gene expression responses to Pb exposure in fish and invertebrates display some consistency. Pb induced expression changes of the above genes are currently being verified by qPCR and will be followed by studies of time and concentration dependency of Pb-induced expression changes.

P081 (SON-1117-567744) Changes in Transcriptome of HepG2 Cells Exposed to Copper: Pathway Mapping and Interactome Identification.
Start time: 8:00 AM
Song, M-O¹, Freedman, J. H.¹, ¹ Duke University, Durham, NC, USA
Copper is an essential trace element that serves as an important catalytic cofactor for cuproenzymes, carrying out fundamental biological functions in growth and development. It has been shown that copper has the potential to generate free radicals and oxidize cellular components through its redox activity. We have previously shown that copper activates transcription through both metal- and oxidative stress-responsive signal transduction pathways. Our hypothesis is that copper modulates the activity of multiple intracellular signal transduction pathways to affect transcription, which ultimately disrupt normal development. The differentially expressed genes after 8-h exposure to copper were grouped by the functional categories of Gene Ontology using Gene Ontology Tree Machine. Significantly enriched GO categories (p < 0.01) in biological process were: response to metal ion, copper ion homeostasis, asparagine biosynthesis and heme oxidation, and those in molecular function were cadmium binding, copper ion binding, transforming growth factor beta receptor binding, asparagine synthase activity and heme oxygenase activity. We also perform mapping of the differentially expressed genes onto biological pathways using GenMAPP, PharmGKB, KEGG and BioCarta using ArrayXPath. Eighty five of the 374 genes (600 M copper) were identified in 17 of the 45 GenMAPP, 5 of the 9 PharmGKB, 26 of the 70 KEGG, and 169 of the 346 BioCarta pathways. Pathways identified included TGF beta signaling pathway, apoptosis, MAPK cascade, glutathione metabolism, p53 signaling pathway, Ras signaling pathway and oxidative stress induced gene expression via nrf2. All these results strongly support our hypothesis. We also identified interactomes with Cytoscape. Interactomes with high module scores have genes such as PKB, SP-1, BAD, STP15 and CD28 as cores. We will perform identification of significant interactomes from transcriptomes with different exposure time and investigation of potential signal transduction pathways based on the identified interactomes.

P082 (BAI-1117-055321) Alterations in gene expression after exposure of mummichogs (Fundulus heteroclitus) to arsenic.
Start time: 8:00 AM
Bain, L¹, Gonzalez, H¹, ¹ University of Texas at El Paso, El Paso, TX, USA
Although arsenic has been detected as a contaminant in water bodies around the world, little is known about its mode of action on both adults and their exposed offspring. We examined changes in the morphology and gene expression in juvenile mummichogs (Fundulus heteroclitus) whose parents were exposed to 172ppb, 230ppb, or 575ppb arsenic for 10 days immediately prior to spawning. Total RNA from 6-week old hatchlings reared in clean water, as well as total RNA from the livers of the exposed adults, was used to examine differential gene expression using a 524-spot cDNA array. In general, the arrays showed an increase in the total number of differentially expressed genes as the concentration increased. Thus, as the concentration was lowered, the fish looked more like control fish with respect to their gene expression profile. There were a number of genes that changed in a dose-responsive manner, including cytochrome c oxidase subunit III, deiodinase, cathepsin C, and a lysozyme precursor, while other genes such as TBT-binding protein, cathepsin C, several ESTs, and MDR were changed in multiple exposure groups. Ultimately, we hope to use these markers to help predict physiological impacts due to arsenic exposure.

P083 (CAR-1117-202124) Discovery of Genes Associated with PCB Toxicity and Resistance in Atlantic Tomcod.
Start time: 8:00 AM
Carlson, E¹, Roy, N², Wirgin, I², ¹ University of Southern Mississippi, Ocean Springs, MS, USA² New York University School of Medicine, Tuxedo, NY, USA
Several populations of fish inhabiting highly contaminated environments possess heritable resistance to early life stage (ELS) toxicity induced by halogenated aromatic hydrocarbons (HAHs) such as TCDD and co-planar PCB congeners. ELS toxicities induced by HAHs in sensitive fish populations include mortality, widespread circulatory failure, edema, and craniofacial /spinal malformations. The molecular mechanisms behind heritable resistance to HAH toxicity in fish populations are unknown. F1 and F2 generation embryos derived from the Hudson River (HR; New York) population of Atlantic tomcod (Microgadus tomcod) are highly resistant to PCB- and TCDD-induced CYP1A expression and ELS toxicity when compared to tomcod embryos of Miramichi River (MR; New Brunswick, CA) and Shinnecock Bay (SB; New York) origin. This study sought to identify novel genes involved in population differences in embryonic response to PCB exposure using custom cDNA microarrays. Microarray probes consisted of 4,416 un-sequenced inserts of randomly-picked clones from a tomcod cardiac cDNA library. Tomcod embryos from three populations (i.e., HR, MR, and SB) were exposed to 2 doses of an environmentally-relevant PCB mixture and screened for dose- and population-specific patterns of gene expression. Greater than 100 clones were found to be significantly altered within each population for each PCB dose, but < 12 PCB-altered clones were shared between any two populations within each exposure group. In addition, <6 significantly-altered clones were shared between high and low PCB exposure groups within any population. Clones displaying significant differences between populations exposed to the high dose of PCB were chosen for identification by DNA sequencing. Of the 28 identified non-ribosomal protein clones, none displayed expression patterns similar to CYP1A. Both cardiac troponin T2 (tnnt2) and cathepsin L precursor appeared to be slightly induced by the high PCB dose in HR embryos and repressed in MR and SB embryos. Interestingly, previous studies have determined that the zebrafish mutant silent heart lacks tnnt2 expression and displays significant cardiomyopathy. Thus, it appears that the PCB-induced gene expression patterns in sensitive tomcod populations may be related to severe cardiovascular dysfunction.

P084 (MUT-1117-825948) p53 family members from mussels: potential biomarkers for oncogenesis.
Start time: 8:00 AM
Muttray, A.¹, Cox, R.², St-Jean, S.³, Reinisch, C.², Baldwin, S.¹, ¹ Department of Chemical and Biological Engineering, University of British Columbia, Vancouver, British Columbia, Canada² Laboratory of Aquatic Biomedicine, Marine Biological Laboratory, Woods Hole, Massachusetts, United States³ Environment Canada, National Water Research Institute, Burlington, Ontario, Canada
Our interest is in the development of DNA array technology for environmental effects monitoring amongst marine species. We have focused on characterization of potentially diagnostic genes, whose expression profiles may be linked to environmental contamination and/or disease onset. Here, we report on novel p53-like tumor suppressor genes in the bivalve molluscs Mytilus trossulus and edulis. These species, widely used for environmental assessment, develop a well-documented condition known as haemic neoplasia (leukemia). The role of p53 and its close relative p73 in onset of molluscan leukemia has been well documented. We previously isolated p53, and now report on two distinct and novel p73 isoforms from the gill of the mussels. The identification of a novel molluscan p73 of this type is particularly relevant to potential oncogenic transformations occurring during onset of leukemia. The nucleotide sequences of the two variants reported here are nearly identical with the exception that one contains a 360 nt truncation in the 5′ coding region. Based on this truncation and concomitant lack of a trans-activation (TA) domain, we designate this novel variant as a DeltaNp73 isoform. While the DeltaN isoform has been well characterized in mammalian species, this report is the first to identify this biologically important p73 isoform in any non-mammalian species. This research further illustrates the excellent utility of the molluscan model for studies involving the molecular mechanisms of oncogenesis in naturally occurring sessile populations. We are interested in the potential roles played by this novel DeltaNp73 isoform during oncogenic transformation in molluscan leukemia. In mammalian species, DeltaNp73 potently inhibits the tumor-suppressive function of p73 and p53, and its over-expression serves as a robust molecular marker for mammalian cancer. With the long-term goal of utilizing p73 gene expression as a molecular marker for oncogenic transformation in marine molluscs, our studies are currently underway to determine leukemia-specific p53, p73 and DeltaNp73 expression, as well as the potential inhibitory functions of the newly identified DeltaNp73 isoform.

P085 (FEN-1117-812890) Gene expression and metabolic profiling in Danio rerio embryos as a strategy for investigating mechanisms of toxicity.
Start time: 8:00 AM
Fenske, M¹, Schaefers, C², Robinson, M¹, Cossins, A³, Maund, S³, ¹ Syngenta, Jealott’s Hill International Research Centre, Bracknell, Berkshire, United Kingdom² Fraunhofer IME, Schmallenberg, Germany³ School of Biological Sciences, University of Liverpool, Liverpool, United Kingdom
Regulators and industry need to develop cost-effective and robust test methods to efficiently predict and assess the potential hazard of chemicals to the environment and human health, while reinforcing animal welfare considerations. Ecotoxicology need to develop approaches, which also allow elucidation of mode-of-action (MOA) and integrate the molecular responses to toxicants. Such integrated approaches may greatly improve our understanding of the mechanisms of toxicity. Applying fish embryos in the development of alternative toxicity (screening) tests offers the opportunity to combine an in-vivo approach with an in-vitro scale test system. The zebrafish (Danio rerio) has emerged as the most suitable species in this context. Apart from being an already well established test species in ecotoxicology it has proved to be a powerful vertebrate model in terms of its reproductive and developmental properties as well as being amenable for forward and reverse genetic approaches. Evidence in the literature of good correlations between acute and zebrafish embryo toxicity data for many toxicants corroborate the potential of this test as an alternative to the acute fish toxicity test. In this project we are developing and validating the zebrafish embryo test as a medium to high-throughput toxicity assay which links morphological and developmental effects with molecular responses at the genome and metabolome level. In a pilot study, embryos were exposed to a selection of environmental toxicants of either known or partially known MOAs at two different concentrations each for 48 h post fertilisation. Embryo development and symptomology were assessed, and surviving embryos after 48 h were subjected to gene profiling analysis using a zebrafish genome array (Affymetrix GeneChip®). We found chemical-specific patterns of altered gene expression although these preliminary results need further verification by single gene expression analysis using real-time PCR. For future studies complementary post-genomic approaches (metabolomics, proteomics) will be included in the analysis.

P086 (KLA-1117-802859) Gene expression in Pimephales and Daphnia spp. after pharmaceutical exposures: links to behavior, survival, reproduction.
Start time: 8:00 AM
Klaper, R¹, Rees, C¹, ¹ University of Wisconsin Milwaukee
Gene expression studies provide a mechanism to examine the functional response of an organism to environmental changes. Through the use of suppressive subtraction libraries, micro- and macro-arrays, differential display, and quantitative PCR, many biomarkers become available to decipher the response of an organism to toxins. Using Pimephales promelas and Daphnia spp. as models, the effects of pharmaceutical (naproxen, clofibrinic acid, and fluoxetene) and methyl mercury exposures were investigated using gene expression. Results demonstrate the potential for genomic markers to be used as an early indicator of physiological response to different chemicals. In addition, gene expression provides an indication of the effects of these chemicals. Pharmaceutical exposure resulted in altered expression of genes associated with chemical specific pathways. Methyl mercury exposure in fatheads results in a decline in reproduction and changes in gene expression associated with reproduction. Mercury reduces vitellogenin expression in female fatheads and increases vitellogenin in male fatheads, but it is a highly variable response. This variation was confirmed using both quantitative PCR and macroarray. Our studies examine the variation in gene expression, sex, and genotype among individuals exposed to the same environment. We have found sex contributes strongly to gene expression response not only in genes associated with reproduction but other functions as well. We have begun to examine the ability to detect differences in gene expression response in the field in naturally occurring populations of fish to determine the capability of using this technique as an early warning of problems due to contaminants.

P087 (BOZ-1117-821883) Embryonic gene expression among natural Fundulus populations: sensitivity and resitance to pollution.
Start time: 8:00 AM
Bozinovic, G.¹, Oleksiak, M.F.¹, ¹ North Carolina State University
Embryos are highly sensitive to pollution, and exposure to contaminated water and sediments can result in altered development and growth and can affect survival. However, while sediment extracts from highly polluted environments can be lethal to Fundulus heteroclitus embryos from clean sites, embryos of parents from the polluted environment are remarkably resistant. What changes during development contribute to this resistance? This study uses microarray analyses to quantify changes in mRNA expression during development in order to test the hypothesis that altered gene expression during sensitive developmental stages contributes to resistance. The patterns of developmentally expressed genes in F1 Fundulus embryos of parents from three independent, geographically unrelated, polluted sites and contrasting populations from clean sites are analyzed. This study addresses following questions: 1. What are the differences in gene expression during sensitive developmental stages between the embryos of fish collected from clean and polluted sites? 2. Which patterns of gene expression are conserved among polluted populations? Conserved differences among populations of Fundulus from polluted sites might indicate common responses to stress and will be pursued for analysis of functional relevance. Differences in developmental gene expression will also be used to target genes and explore mechanisms that underline biological responses caused by chronic exposure to pollutants.

P088 (WAN-1117-816850) CYP1 mRNA expression in Fundulus and catfish following BaP Exposure.
Start time: 8:00 AM
Wang, L¹, Scheffler, B², Ford, A¹, Ganesan, S¹, Willett, K¹, ¹ University of Mississippi, University, MS, USA² USDA-ARS, Stoneville, MS, USA
While CYP1A has been recognized, studied and used as a biomarker in fish for many years, understanding of the significance of more recently cloned fish CYP1B and CYP1C genes is lacking. In mammals, CYP1B1 is responsible for activating polycyclic aromatic hydrocarbons, such as benzo(a)pyrene (BaP) and estradiol to carcinogenic intermediates. We have cloned full length and partial CYP1-like cDNAs from the channel catfish and Fundulus heteroclitus, respectively. The channel catfish CYP1B codes for a putative protein of 536 AA that shares the highest amino acid identity with carp 1B2 (68%). Fundulus partial cDNA was most similar to scup 1C1 (66%). Compared to mammals, the physiological significance, metabolic activities and inducers of teleost CYP1s apart from CYP1A are largely unknown. We found CYP1B mRNA was detectable in catfish gill, liver, gonad and blood and statistically significantly inducible by 20 mg/kg i.p. BaP exposure. In dose-response studies with catfish primary cultured gill cells, the tissue with the highest constitutive levels of CYP1B, BaP, TCDD, PCBs 77, 126, and 169 induced CYP1B message by 10-fold or more relative to DMSO controls. On-going studies are using Fundulus embryos to investigate the developmental expression of CYP1s following exposure to waterborne BaP (10 g/L) for the initial 10 days postfertilization. Our results suggest that teleost CYP1s, in addition to CYP1A, are inducible by AhR agonists, have a broad tissue distribution, and may be physiologically significant biomarkers of contaminant exposure.