Fent K - Progress and Promises Toxicogenomics in Aquatic Toxicology - Aquatic Toxi - 2012

Aquatic Toxicology 105S (2011) 2539
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Aquatic Toxicology
journal homepage: www.elsevier.com/locate/aquatox
Progress and promises in toxicogenomics in aquatic toxicology: Is technical innovation driving scientic innovation?
Karl Fent a,b, , John P. Sumpter c
a b c
University of Applied Sciences Northwestern Switzerland, School of Life Sciences, Grndenstrasse 40, CH-4132 Muttenz, Switzerland Swiss Federal Institute of Technology (ETHZ), Department of Environmental Sciences, CH-8092 Zrich, Switzerland Brunel University, Institute for the Environment, Uxbridge UB8 3PH, United Kingdom
a r t i c l e
i n f o
a b s t r a c t
In the last decade, new technologies have been invented to analyze large amounts of information such as gene transcripts (transcriptomics), proteins (proteomics) and small cellular molecules (metabolomics). Many studies have been performed in the last few years applying these technologies to aquatic toxicology, mainly in sh. In this article, we summarize the current state of knowledge and question whether the application of modern technology for descriptive purposes truly represents scientic advancement in aquatic toxicology. We critically discuss the advantages and disadvantages of these technologies and emphasize the importance of these critical aspects. To date, these techniques have been used mainly as a proof of principle, demonstrating effects of model compounds. The potential to use these techniques to better analyze the mode-of-action of a toxicant or the effects of a compound within organisms has rarely been met. This is partly due to a lack of baseline data and the fact that the expression of mRNA and protein proles is rarely linked to physiology or toxicologically meaningful outcomes. It seems premature to analyze mixtures or environmental samples until more is known about the expression proles of individual toxicants. Gene transcription, protein, or metabolic data give only a partial view of these effects. Thus, we emphasize that data obtained by these technologies must be linked to physiological changes to fully understand their signicance. The use of these techniques in aquatic toxicology is still in its infancy, data cannot yet be applied to environmental risk assessment or regulation until more emphasis is placed on interpreting the data within their physiological and toxicological contexts. 2011 Elsevier B.V. All rights reserved.
Keywords: Transcriptomics Gene expression analysis Fish Proteomics Metabolomics Critical review
1. Introduction This article presents an overview of the past (over the last decade) and presents developments in new technologies introduced in the eld of aquatic toxicology. We critically discuss the state of the art with a focus on sh, and compare the proposed benets of these new technologies with the progress made in the eld today. We also explore the limitations of these technologies, as well as provide a perspective on possible future developments in this eld. 2. Toxicogenomics in aquatic toxicology The advent of new technology often has great potential. The sequencing of the genome of the zebrash (Danio rerio) and the Japanese puffersh (Takifugu ribripes) has fostered the develop-
Corresponding author at: University of Applied Sciences Northwestern Switzerland, School of Life Sciences, Grndenstrasse 40, CH-4132 Muttenz, Switzerland. Tel.: +41 61 467 4571. E-mail address: karl.fent@fhnw.ch (K. Fent). 0166-445X/$ see front matter 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2011.06.008
ment of microarrays for use in biology, development biology, and aquatic toxicology. Currently, genomic information in these sh species, as well as in Japanese medaka (Oryzias latipes) and fathead minnows (Pimephales promelas), nds most frequent application in aquatic toxicology. Forty complete genome sequences are available, 409 species are assembled and further 665 species sequencing is in progress (www.ncbi.nlm.nih.gov/genomes/leuks.cgi). Also important for aquatic toxicology are the entire genome sequences of zebrash, the water ea Daphnia pulex (http://wFleaBase.org) (Colbourne et al., 2011), and the algae Chlamydomonas reinhardtii, Chlorella variabilis NC64A, as well as the frog Xenopus tropicalis. The assembly of genomes of 15 sh species is listed, and sequencing is also in progress for a further 22 species (www.ncbi.nlm.nih.gov/genomes/leuks.cgi). Sequence data have fostered research in many areas including toxicology and aquatic toxicology. Among other applications, including primer design for quantitative PCR, genomic information can be used to design and develop microarrays for some selected genes, thousands of genes, or even almost the entire genome (e.g. zebrash). Microarrays are constructed by cDNAs or oligonucleotides and spotted arrays are commercially available for a few sh species. In cases where a large set of genes or
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K. Fent, J.P. Sumpter / Aquatic Toxicology 105S (2011) 2539
almost the entire genome is assessed, the term global expression analysis is used. Microarrays can be used to determine upregulation and downregulation of gene transcripts (mRNA) in cells, tissues, organs, or even the entire organism in specic physiological conditions (e.g. environmental stress such as hypoxia), development (embryogenesis), or exposure to an environmental chemical, chemical mixtures, or environmental samples. The term transcriptomics is used when transcriptional changes in exposed organisms are described. The combination and advances in DNA sequencing and the development of microarray technology made it possible to analyze transcription of large sets of genes and to obtain an unanticipated quantity of data. In addition, heterologous applications of microarrrays have been made, meaning that arrays constructed of oligononucleotides of known sequences of a sh (e.g. zebrash) are used for other species of sh. For instance, heterologous applications include the information from puffersh used for rainbow trout (Hogstrand et al., 2002), salmon microarray (Von Schalburg et al., 2008; Rise et al., 2004), European ounder (Diab et al., 2008; Cohen et al., 2007), carp (Williams et al., 2008) and catsh (Liu et al., 2008). Rise et al. (2004) demonstrated heterologous application of the salmon dDNA microarray even for distantly related sh. Of course the data strength is lowered by the use of heterologous arrays, and in general it is desirable to use species-specic microarrays.
Box 1: Applications of toxicogenomics, proteomics and metabolomics data. as a test of principle and proof of concept to describe and understand the modes of action of a compound to predict modes of action and effects of a compound based on the proles to discover and describe new biomarkers of effect (Lam et al., 2008; Gunnarsson et al., 2007) to perform effect assessments: environmental chemicals with known modes of action. environmental chemicals with partly or completely unknown modes of action. compound mixtures. efuents and environmental samples. to distinguish between organisms living in polluted versus unpolluted environments. as a diagnostic tool to determine environmental contamination, and also efcacy of pollution remediation (Roling et al., 2007). to identify effects induced by complex mixtures (GarciaReyero et al., 2008; Filby et al., 2007)
2.1. Perspectives and possibilities 2.1.1. Hypotheses One of the basic hypotheses behind toxicogenomic is that alterations in gene transcripts represent the primary action between chemicals (xenobiotics) and biota; it represents the rst action prior to the onset of physiological and biological changes. Also, environmental factors (temperature, pH, anoxic conditions, etc.) may directly affect transcription. Targeted transcript expression analyses of genes using quantitative real-time polymerase chain reactions (qRT-PCR) allow the determination of changes in transcript expression of a dened and selected series of genes. This approach is straightforward and allows analysis of known or suspected expressional changes based on the mode of action of the compound, as for instance the endocrine activity of UV-lters (Zucchi et al., 2011a,b). However, a limitation of this approach is that only a small set of genes can be analyzed, whereas microarrays offer the opportunity to analyze thousands of genes or almost the entire genome. Global approaches using microarrays that measure almost all of the genes transcribed at a specic time and at specic concentrations are assumed to be superior, as the toxicity of chemicals involves mostly multiple modes of actions and a cascade of gene interactions and pathways rather than the changes in transcript expression of only a few genes. Therefore, one of the possible advantages of toxicogenomics is that microarray technology may enable (in a holistic or global approach) us to analyze all of the expressional changes in an organism or tissue. Hence, it is thought that all relevant expressional changes can be discovered at a given point and at a given concentration, like a snapshot in time. It is assumed that this will allow identifying the modes of action of the compound and associated adverse effects. Additionally, it is hypothesized that, based on the mode of action, a specic expressional prole will result in a transcriptional ngerprint of the compound. A comparison between the expressional proles and the ngerprints of known model compounds will then form the basis for identifying compounds with a similar mode of action. It can also be used to identify the compounds action in mixtures and in environmental samples. Therefore, a compound can be identied by its specic mode of action or affect on a specic organ in an
animal (e.g. brain, liver). This can be used to identify the potential adverse effects of a compound based on the transcriptional prole. Ultimately, we hope to identify and describe a pattern of expressional changes for a given mode of action. The basis of this hypothesis is that microarray-based experiments will reveal a mode of action prole as well as identify a link between transcript expression proles of genes and the mode of action. Changes in transcript expression are hoped to be toxicant-specic and to be linked to a mode of action for that chemical. Therefore, different compounds can be compared to determine if those compounds have similar modes of action. Furthermore, it is hypothesized that alteration in transcript expression of genes translates into physiological alterations. Transcriptional proles can than be used to identify toxic effects. Similarly to toxicogenomics, new technology focused on the protein expression pattern (proteomics) and presence of metabolites (metabolomics) in conjunction with bioinformatic tools have the potential to discover the protein and/or metabolite alterations indicative of the mode of action of chemicals. They could improve our understanding of mechanisms of action of chemicals as well predict changes via the comparison of chemicals sharing the same mode of action. Currently, all of these techniques remain in the domain of research. The application of these methods in environmental risk assessment of chemicals and environmental samples is certainly premature. Data usage is listed in Box 1 . 2.1.2. Some complications To complicate the issue, most environmental chemicals have not only one but a series of molecular effects, or their effects vary in time and with concentration. There are genes responding within a short period of time (hours), others that are only differentially regulated after longer terms of exposure, or transiently expressed. Some genes will probably be more sensitive than others, so at low concentrations of a chemical, only a reduced suite of genes may be affected. At higher concentrations, a much larger suite of genes may well be affected. Very high concentrations of a chemical may be toxic and lead to other genes being affected as a consequence of the cellular damage. There does not need to be a correlation between the magnitude of expressional changes, responsiveness or the expressional time prole. For chemicals with unknown or
K. Fent, J.P. Sumpter / Aquatic Toxicology 105S (2011) 2539 Table 1 Advantages and disadvantages of microarray studies.a Advantages Unbiasedtranscript expression of every gene can be theoretically determined (both in a tissue or in whole organism). Gives the whole picture on potential effects of a compound rather than merely a small part of it (if whole genome microarrays are used). In theory, transcript expression can be traced to protein synthesis level, and even metabolism (if all three omics were to be included in one study). Completely unexpected pathways and mechanisms of action can be discovered. Large amounts of data can be obtained allowing new hypotheses to be generated and explored. In theory, an expression prole typical of a specic activity (e.g. estrogenic) can be identied. Very expensive (for both hardware and consumables and bioinformatics tools). Required skilled and trained researchers, which are often lacking, both in microarray techniques, bioinformatics, and data interpretation. Transcript expression levels of genes (in response to a toxicant) are time-dependent and concentration-dependent. Thus, important time points may be missed. Interpretation of results is often highly challenging and difcult. Many genes are not annotated and their functions remain unknown. Currently, the use of diverse techniques is not fully developed or validated. Standardization of data collection and analysis are lacking. Often provides a shotgun approach, rather than a hypothesis-driven approach, to research. May not provide any added value to the targeted transcript expression approach using qRT-PCR. Use of heterologous microarrays (application of a microarray of one species to another species) is problematic. Incomplete genome data for many species restrict use of microarrays. Transcript expression data give only a partial view on the effects; they must be linked to physiological changes. Transcriptomics is in its infancy, data cannot yet be applied in environmental risk assessment or regulatory context.
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Disadvantages
a Although this table covers the advantages and disadvantages of the use of microarray technology to reveal transcript expression patterns of genes, very similar tables, with many of the same points, could be constructed for both proteomics and metabolomics.
widely dispersed effects, interpretation of data from these new technologies becomes problematic. Table 1 lists some of the important benets and limitations of transcriptomics. 2.1.3. Predictive approaches: the hypothalamicpituitarygonadal axis In general there are only a few examples where a transcript expression prole of a compound with a certain mode of action has a predictive value for other unknown compounds. It is hoped that a ngerprint can be found for estrogenic (Kausch et al., 2008), androgenic, antiandrogenic and aromatase-inhibiting (Villeneuve et al., 2007) compounds so that they can be used to identify similarly acting compounds. Thus far, the data are not very promising, but rather demonstrate that we have a long way to go to truly fulll the potential of the predictive power of expression patterns. A graphical system model was proposed by Villeneuve et al. (2007) to predict effects of endocrine active compounds on the signaling along the hypothalamicpituitarygonadal (HPG) axis in sh by utilizing existing literature data and by testing the aromatase inhibitor fadrazole in fathead minnows. Not all predicted changes were observed, perhaps partly because the targeted microarray that was used focused on a limited and selected gene set. As a kind of summary evaluation based on different studies, recently, a concept of adverse outcome pathway has been proposed to forge a mechanistic link between transcript expression and effects by focusing on ve case examples (Ankley et al., 2010). It was proposed that some genes are associated with the initiating events that, via a cascade of biochemical interactions, lead to a predictable adverse outcome. The effects of chemicals acting on the estrogen receptor, ultimately causing adverse effects on reproduction, are certainly a good case. However whether this is enough to predict impacts of chemicals on organisms or even populations is uncertain. Yet, another concept has been proposed by the same authors: a transcriptome-based framework of signal transduction pathways and transcriptional regulatory networks on the HPG axis (Wang et al., 2010a,b). In zebrash, four tissues of both genders were analyzed for their transcriptional changes upon various exposures to EE2, fadrazole, trenbolone, pronil, prochloraz, utamide, vinclozoline, muscimol, ketoconazole, and trilostane. Analysis revealed that the pathways and transcription factors showed interactions among the HPG axis. Impacts on cellular functions, such as stress response, cell cycle, and apoptosis, were common features. This synthesis work on several studies of these authors aimed to facilitate hypotheses regarding the mode of action of endocrine dis-
Box 2: Some current achievements of toxicogenomics. Proof of principle in that known genes are altered as expected (e.g. vitellogenin induction). Knowledge that transcriptional effects of a chemical are dose-dependent and time-dependent. In addition to molecular pathways other instances of alteration of genes and pathways were discovered. Elucidation of novel modes of action of toxicants. In addition to expected molecular pathways, alteration of other genes and additional pathways were discovered. Knowledge that a single chemical can affect hundreds up to thousands of genes. The discovery that alterations of transcript expression of genes may be more sensitive than physiological endpoints and indicative for physiological changes. They may appear more problematic than physiological outcomes might reveal them to be.
rupters in sh. The authors also tried to identify novel biomarkers for some of these compounds (Wang et al., 2008). Yet to date, only a few studies have been performed that link changes in transcript expression of genes to physiology or responses at higher biological levels. Attempts have been made to explore the links between gene transcript expression responses and responses at the population level (Soetaert et al., 2007; Connon et al., 2008) in Daphnia magna upon exposure to cadmium. Changes in transcript expression of many genes caused by exposure to cadmium seem to be linked to somatic growth, development, and (consequently) population growth rate. Some recent achievements are listed in Box 2 , and Table 1 lists some of the important advantages and disadvantages of microarray studies. Box 3 lists some important prerequisites, such as baseline data. Currently, relatively little is known about the key factors inuencing gene expression patterns. Put another way, the key baseline data ideally required for optimal experimental design, appropriate analysis for results, and correct interpretation of those results, are currently missing. Microarray technology is presently being used, for example, to investigate the toxicity of a chemical within the necessary baselines being established. The difculties this produces can be readily illustrated by considering the use of plasma vitel-
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Box 3: Key baseline data required for optimal experimental design and data interpretation. Intra-sh variation in expression patterns. The inuence of the following factors needs to be established: Temporal changes (i.e. how do gene expression responses change with time?). Changes during ontogeny (e.g. differences between juveniles and adults). Changes due to sexual maturation and reproduction. Inter-sh variation in expression patterns: Differences between males and females. Differences between species. Effects of environmental conditions on expression patterns: Effects of stress. Effects of feeding. Effects of light/dark cycle (including circadian rhythmicity of gene expression).
(Williams et al., 2003). Since these early studies, toxicogenomics has been applied in many different organisms, from algae to sh. Of course, data derived from homologous microarrays have more power than heterologous systems because of a clearer assignment of gene products. Gene expression analysis can be used to determine the transcription prole of a compound acting via a selected pathway. Subsequently this information, or the compounds ngerprint, is thought to be used to determine (1) whether compounds with unknown modes of action follow a given mode of action prole, (2) whether compounds in a mixture or in environmental samples have a similar mode of action. In the following section different approaches are critically discussed. 2.2.1. Compounds with known modes of action (model compounds) 2.2.1.1. Metals, metallic nanoparticles and organometals. So far, almost all environmental chemicals analyzed by microarrays are chemicals known for their mode of action, both in sh and invertebrates. In Daphnia, for instance, the effects of metals have been analyzed (Poynton et al., 2007). The toxicity of metals is largely dependent on speciation, and therefore on water chemistry. This has also been found by expressional patterns of metals that are inuenced by chelators present in water. D. magna microarrays were applied to identify expression proles in response to copper, cadmium, and zinc exposures. Metallothioneins and ferritin mRNA were identied (Poynton et al., 2007). The mRNA expression patterns supported known mechanisms of metal toxicity. Additionally, in Daphnia, mRNA expression of several genes was assayed after short-term metal exposure (Vandenbrouck et al., 2009). Microarray analysis of nickel-exposed zooplankton revealed several affected functional gene classes, which were predicted to be involved in different metabolic processes, cuticula turnover, transport, and signal transduction. Furthermore, transcription of genes involved in oxygen transport and heme metabolism was downregulated. An inverse relationship was observed between the mRNA expression levels of different cuticula proteins and available energy reserves. In addition to the nickel exposure, exposure to binary mixtures of nickel and cadmium, or nickel and lead, were analyzed, revealing a complex interaction. The transcriptional response of uranium was studied in the brain of the zebrash, exposed to 15 and 100 g/L for 3 and 10 d (Lerebours et al., 2010). A total of 56 transcripts responded to uranium exposure, and the anatomical structure of the olfactory bulb was damaged. Gene response was higher at the lower concentration and the numbers of responding genes common to any two exposures were much smaller than those unique to each exposure. These data showed that the intensity of gene response may not correlate with concentrations. Instead, different patterns of mRNA expression of genes occurred for each exposure concentration. Gene responses were categorized into eight functional classes, and the transcriptional responses of genes involved in the olfactory system were signicantly affected. The acute toxicity of soluble copper and 80 nm copper nanoparticle suspensions were examined in zebrash (Griftt et al., 2007). Histological and biochemical analysis revealed that the gill was the primary target organ for nanocopper. Nanocopper produced different morphological effects and global transcript expression patterns of genes in the gill compared to soluble copper. Effects of dietary uptake of methylmercury (MeHg) were studied in fathead minnows, Pimephales promelas, and the mRNA expression of genes commonly associated with endocrine disruption were altered (Klaper et al., 2006). Upregulation of vitellogenin mRNA in males and downregulation in female sh was observed. Additional genes identied included those associated with egg fertilization and development, sugar metabolism, apoptosis, and
logenin concentrations as a biomarker for estrogenic chemicals; 25 years after the variation in plasma vitellogenin concentrations in sh, during the reproductive cycle, were rst documented (Copeland et al., 1986) concentrations in normal male sh (the control value in most ecotoxicological experiments) is still being debated (Beresford et al., 2011). And if the control value is not known then it can be impossible to know if the concentrations in experimental sh (in laboratory tests of chemicals, or wild sh exposed to contaminated water) are, or are not, abnormal. The outcome then varies from unhelpful research to results that cannot be replicated. Of course, it is not necessary to have all the baseline data available before ecotoxicological experiments utilizing microarrays are conducted, as long as the lack of baseline data is accommodated within the experimental design. For example, it is not necessary to know how gene expression patterns vary between males and females if only one sex is used in an experiment. Nor is it necessary to know how sexual maturation and reproduction effects gene expression patterns if only sexually immature sh are used in a study. However, the inuences of other factors are much more difcult to exclude, irrespective of how an experiment is designed. For example, in response to a challenge by a chemical, the gene expression pattern changes dramatically with time: the genes affected after 2 h will be completely different from those effects displayed after 12 or 24 h (Moggs, 2005). Similarly, it is likely that the gene expression changes in the liver induced by, for example, a reproductive toxicant will be radically different to those occurring simultaneously in the brain, pituitary gland, and gonad. Hence, not knowing how gene expression is affected by both internal (e.g. sexual maturation) and external (e.g. stress, feeding) factors, and how tissue-specic those gene expression patterns are, can make it difcult to conduct a meaningful experiment and interpret the data. 2.2. Applications Over the last decade, toxicogenomics has found application in aquatic toxicology in various ways. In early studies its feasibility has been demonstrated by using low-density microarrays (Larkin et al., 2003), characterizing the response to endocrine disrupters. Additionally, genomic information from the puffersh has been applied for studying the response of rainbow trout to zinc (Hogstrand et al., 2002). At an early stage, microarrays have also been used to distinguish contaminated from pristine environmental sites by hepatic gene expression proles in the European ounder Platichtys exus
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electron transport. MeHg differed in its effect from other metals or compounds known as endocrine-disrupters. The analysis of muscle tissue after MeHg exposure results in a different expression prole (Cambier et al., 2010). Perturbation of protein synthesis, mitochondrial metabolism, endoplasmic reticulum function, detoxication, and general stress responses were predicted to be differentially regulated in exposed zebrash. Several other cellular genes had differential mRNA expression, such as those predicted to be involved in lipid metabolism, calcium homeostasis, iron metabolism, muscle contraction, and cell cycle regulation. A schematic representation of the general cellular response caused by MeHg contamination demonstrates that many alterations were not common to both muscle and brain tissues. This clearly shows that expressional patterns differ between tissues. 2.2.1.2. Endocrine disrupters. Most environmental chemicals studied so far include organic chemicals acting as endocrine disrupters and metals. Most toxicogenomics studies focused on chemicals acting on steroid signaling in sh, namely estrogens (17-b-estradiol (E2), 17-a-ethinylestradiol (EE2)), androgens (17a-methyldihydrotestosterone, dihydrotestosterone), synthetic androgens (trenbolone), antiandrogens, and the aromatase inhibitor fadrazole. Chemicals acting as endocrine disrupters (bisphenol A, nonyphenol) have also been studied. Investigated tissues include the hypothalamus (Martyniuk et al., 2006; Marlatt et al., 2008), brain (Martyniuk et al., 2007), liver (Moens et al., 2007; Benninghoff and Williams, 2008; Hoffmann et al., 2006, 2008), and gonads (Santos et al., 2007). Transcriptomic studies have also been performed on whole zebrash (Lam et al., 2008). Often environmental compounds do not only act via one but multiple modes of action, which as a consequence can cause responses that are phenotypically identical. For example organochlorine pesticides having additional effects besides endocrine disrupting effects were analyzed in M. salmoides (GarciaReyero et al., 2006). Thereby, three pathways of endocrine disruption were analyzed (receptor interaction, changes in hormone synthesis, changes in hormone metabolism), and transcriptional alterations occurred in all pathways. Lam et al. (2008) used whole-body zebrash transcriptomics to evaluate its potential for predictive and discovery purposes by focusing on two classes of important compounds, Ah-receptor agonists such as PAHs and dioxins (represented by benzo[a]pyrene, 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzodioxin) and estrogenic compounds (represented by E2, diethylstilbestrol and bisphenol A). The data indicate that these two compound classes have different transcriptomic proles. The authors derived prediction models that can differentiate between compounds of the same class and those of different classes in two large independent experiments. The expression signatures also led to the identication of biomarkers for potent aryl hydrocarbon receptor (AHR) and estrogen receptor (ER) agonists, respectively, and were validated in multiple targeted tissues. 2.2.1.3. Estrogens. Several studies analyzed the effects of E2 in sh (Lam et al., 2008; Larkin et al., 2002; Tilton et al., 2006). Williams et al., 2007) evaluated the transcriptional prole in European ounder, Platichthys esus, to E2. Known biomarkers of estrogen exposure, such as choriogenin L and vitellogenins, showed sustained transcriptional induction. Among 175 identied genes, mitochondrial genes, those involved in amino acid synthesis, ubiquitination and apoptosis were transcriptionally induced while those associated with immune function, electron transport, cell signaling and protein phosphorylation were transcriptionally repressed. Additionally, several studies in different sh analyzed transcript expression proles caused by EE2 (Martyniuk et al., 2007;
Filby et al., 2007; Wang et al., 2010a,b). Furthermore, the expression proles of male zebrash exposed to different estrogens were compared (Kausch et al., 2008); the expression proles in response to exposure to bisphenol A did not resemble those from EE2 or genistein. Only well known vitellogenin and zona radiata protein transcripts were regulated in common among the three compounds, of which bisphenol A caused the fewest expressional changes. This example demonstrates that mainly already known toxicological effects of estrogens were identied, thus being conrmative rather than complementary to previous knowledge. 2.2.1.4. Androgens and antiandrogens. Exposure of rainbow trout to trenbolone resulted in altered mRNA expression of 64 genes, far fewer than the number observed in rainbow trout following exposure to EE2 (Hook et al., 2006). Further, a greater proportion of genes were transcriptionally upregulated following exposure to trenbolone, whereas more genes were transcriptionally downregulated following exposure to EE2. The androgenic compound trenbolone and the antiandrogen utamide have been extensively studied in sh by microarray studies. Garcia-Reyero et al. (2009) studied these compounds in fathead minnow ovaries as single chemicals and as a mixture. Flutamide altered about twice the number of genes as trenbolone, most of which were not associated with the action via the androgen receptor. The antagonistically acting compounds resulted in 37 genes that were assigned to human homologs which were reciprocally regulated by trenbolone and utamide. Out of these, the authors suggest that some could be used as potential biomarkers for androgenic or antiandrogenic activity. The microarray study revealed that many different genes and pathways were altered, revealing a complex effect pattern that cannot be easily resolved. Moens et al. (2006) examined the transcriptional effects of endocrine disrupters with different modes of action, where utamide did not group closely with vinclozolin, another model antiandrogen, but instead appeared to be more closely linked to the effects of T3 (triiodothyronine), T4 (thyroxine), and dibutylphthalate. Once again, this study demonstrates a complex pattern of affected pathways. Gene transcript expression proles of the androgen 11ketotestosterone (11-KT), the antiandrogen utamide, and the antiandrogenic fungicide vinclozolin were studied in medaka (Oryzias latipes) larvae (Leon et al., 2008). Flutamide, vinclozolin, and 11-KT caused signicant differential transcript expression of at least 87, 82 and 578 genes, respectively. Two sets of responsive genes are associated to vertebrate sex differentiation and growth, and 50 genes were useful in discriminating between the compounds. The discriminating capacity was conrmed by the similarity of the antiandrogenic expression proles of vinclozoline and utamide, which were distinct from the androgenic prole of 11K. Therefore, this study contradicts the ndings of Moens et al. (2006), where utamide and vinclozolin apparently caused different expression proles in fathead minnows. Hepatic gene expression proling was performed in female zebrash exposed to 17a methyl-dihydrotestosterone (MDT) for 24 and 168 h at three concentrations (Hoffmann et al., 2006). Exposure to MDT resulted in an increase in plasma E2 while plasma levels of testosterone were reduced. A rather small number of gene transcripts, involved in a variety of biological processes, were altered. Genes involved in retinoic acid metabolism, steroidogenesis and steroid metabolism, hormone transport, and regulation of cell growth and proliferation were affected. In the liver, 171 genes were affected in a concentration-dependent manner. Genes identied in this study provide information on the potential mode of action of strong androgens in female sh, but the mode of actions of MDT could not be identied.
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2.2.1.5. Organophosphates. Organophosphates such as dithiocarbamates (DTC) are widely used in pesticides and residues are found in the environment. DTC are found to be teratogenic to vertebrates, but the mechanisms are not well known. Tetraethylthiuram (thiram), tetramethylthiuram (disulram), and sodium metam (metam) lead to craniofacial abnormalities in developing zebrash embryos (Van Boxtel et al., 2010). In order to better understand the molecular mechanisms underlying DTC teratogenesis, embryonic zebrash (PAC2) cells were exposed to thiram and disulram and changes in transcript expression were analyzed by microarrays. The transcript expression of 166 genes that were specic for exposure to DTC was reported, and a network of genes was identied which were related to connective tissue development and function. Additionally, eight transcriptionally downregulated genes related to transforming factor b-1 (TGF-b1) signaling were identied, including an essential transcription factor for zebrash craniofacial development, SRY-boxcontaining gene 9a (sox9a). The authors also showed that sox9a transcript expression is altered in the ceratobranchial arches of DTC-exposed zebrash. Therefore the microarray data suggest that this is an important event in the development of DTC-induced craniofacial abnormalities. By the use of microarrays, this study demonstrates its usefulness in elucidating the molecular basis for teratogenicity of such compounds, and in gaining a better understanding of DTC-induced teratogenesis in vertebrates. 2.2.1.6. Pharmaceuticals. Effects of different concentrations of mianserin on zebrash (Danio rerio) transcript expression using a brain-specic microarray have been studied (Van der Ven et al., 2006). Similarly, the impact of exposure on egg production, fertilization, and hatching has also been assessed. After 2 d of exposure microarray analysis showed a clear effect of mianserin on important neuroendocrine-related genes (e.g., aromatase and estrogen receptor), suggesting that antidepressants can modulate neuroendocrine processes. Adverse effects on egg viability were seen after 14 d of exposure at the highest concentration. We investigated the effects of the benzodiazepine diazepam (ValiumTM ), a known sedative in mammals, by whole genome microarrrays in the brain of zebrash (Oggier et al., 2010). Transcriptomics was used to identify molecular effects of diazepam and to discover its neurotoxic mode of action combined with neurobehavioral studies and thus it was possible to relate expressional changes with physiology. Male zebrash and zebrash embryos were exposed for 14 d or up to 3 d after hatching, respectively, to determined water concentrations of 235 ng/L and 291 g/L. Among the 51 and 103 altered transcripts at both concentrations, respectively, the transcript expression of genes involved in the circadian rhythm in adult zebrash and eleuthero-embryos were of particular signicance, as revealed both by microarrays and quantitative PCR (Fig. 1a). The microanalysis showed a striking effect on the circadian rhythm not previously described. Therefore, toxicogenomics led to the identication of a new mode of action of this environmental toxicant. The swimming behavior of eleuthero-embryos was signicantly altered at 273 g/L (Fig. 1b). The study leads to the conclusion that diazepam-induced alterations of genes involved in circadian rhythm are paralleled by the effects in neurobehavior at high, but not at low, diazepam concentrations. Environmental concentrations, however, lead to expressional changes. To identify molecular effects of the antineoplastic agent, protein kinase C inhibitor 412 (PKC412) (midostaurin), we applied gene expression proling to zebrash using whole-genome microarrays (Oggier et al., 2011). Behavioral, developmental, and physiological effects were investigated in order to identify correlations between altered transcript expression proles with effects on development and physiology. Zebrash embryos were exposed for 6 d postfertil-
ization to nominal levels of 2 and 40 g/L PKC412. Among the 259 and 511 altered transcripts at both concentrations, respectively, the transcript expressions of genes involved in the circadian rhythm were further investigated (Fig. 2a). No alteration in swimming behavior was observed. The pathways affected by PKC412 were angiogenesis, apoptosis, DNA damage response, and response to oxidative stress. Angiogenesis was analyzed in double-transgenic zebrash embryos but no major defects occurred. Apoptosis occurred in olfactory placodes of embryos exposed to 40 g/L, and DNA damage was induced at both PKC412 concentrations (Fig. 2b). However, there were no signicant effects on reactive oxygen species formation. This study demonstrates that PKC412-induced alterations of gene transcripts are partly paralleled in physiological effects at high, but not at low, PKC412 concentrations. Additionally, this study demonstrates the complex expressional pattern of chemicals on the transcriptome and the importance of linking expressional proles with physiological effects. The aromatase inhibitor fadrozole was investigated in several studies. Villeneuve et al. (2009) reported on global transcript expression proles after exposure to 25 or 100 g/L in the brain and ovaries of zebrash after 24, 48 and 96 h. During the study, hundreds of gene transcripts were altered. Based on the transcriptional changes, it was hypothesized that fadrozole induces neurodegenerative stress in the brain. In the ovary, the affected biological processes produced transcript expression changes different from those of the brain. They were functionally linked to cellcell adhesion, extracellular matrix, vasculogenesis, and development. It was also hypothesized that changes in gene expression in the ovary indicate decreased oocyte maturation and ovulation because of impaired vitellogenesis. These hypotheses, derived from the microarray results, provide a basis to test for other similarly acting compounds. Again this study shows the complex transcription prole of a chemical as well as its transcriptional responses, revealing signicant differences at different times and concentrations. 2.2.1.7. Additional compounds. Tilton et al. (2008) analyzed peruoroactanoic acid (PFOA) in rainbow trout for its tumor promoting activity. Extremely high concentrations of PFOA, and another tumor promoter, dehydroepiandrosterone, resulted in estrogenic gene signatures with strong correlation to E2, whereas clobrate had no regulated genes in common with E2. The data suggest that the tumor-promoting activities of PFOA in trout are due to novel mechanisms involving estrogenic signaling and are independent of peroxisome proliferation. Gene expression proles of cyanobacterial toxins for the early life stages of zebrash were analyzed, both as extracts and pure microcystin-LR (Rogers et al., 2011). Changes in global gene expression were also evaluated. The number of differentially transcribed genes increased with MC-LR concentration and included genes related to known mechanisms of action for MC-LR. Transcriptional upregulation of vitellogenin was observed in Microcystis-exposed larvae but not in larvae exposed to MC-LR, suggesting that Microcystis may be a source of environmental estrogens. A summary of insights obtained with model compounds is given in Box 4 . 2.2.2. Compounds with unknown or partly unknown modes of action Model compounds having a dened toxicological prole demonstrate a complex expression prole. The expressional prole may eventually be used to predict the toxicological properties of a given compound. Hence, it is believed that a specic expressional pattern can be applied to differentiate between compounds of the same toxicological class and those of different classes (Lam et al., 2008). However, the identication of toxicological pathways of compounds with unknown toxicological features is even more challenging. Similar to the way in which the net ecotoxicological
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Fig. 1. Comparison of the level of transcript expression of selected genes of the circadian rhythm (a) in zebrash brain determined by microarray, qRT-PCR (zebrash brain and eleuthero-embryos, respectively), and locomotor activity (b) of zebrash eleuthero-embryos exposed to 273 ng/L and 273 g/L diazepam. Many genes are altered dosedependently. Alterations found by microarrays (white bars) are conrmed by qPCR in both adult brain (grey bars) and eleuthero-embryos (black bars). As a consequence of transcriptional alterations of genes of the circadian rhythm, a measurable physiological outcome, neurobehavior, is changed by diazepam. The locomotor activity increased (which seems paradoxically, but this is also seen in humans (children) and animals after diazepam treatment). (a) Values at both diazepam concentrations derived from microarrays (14 d exposure, n = 4 replicates, 15 male sh pooled, white bars), qRT-PCR in brain of adults (n = 5, 15 male sh pooled, grey bars, and in zebrash eleutheroembryos (n = 6, 15 pooled, black bars) are expressed as average fold change (log2 ) with standard error compared to controls. Locomotor activity (percent of total time spent in locomotion during embryos 2 h) of eleuthero-embryos exposed for up to 3 d after hatching. *, statistically signicant difference to control (p < 0.05). Adapted from Oggier et al. (2010) and Oggier et al. (2011).
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Fig. 2. Effects of protein kinase inhibitor PKC412 on transcript expression (a) in zebrash eleuthero-embryos determined by microarray, and DNA damage determined by Comet assay (b). Embryos (n = 4 replicates, 80 eleuthero-embryos pooled) were analyzed after exposure to 1.3 g/L and 21 g/L PKC412, respectively, in the microarray experiment (a), and to 1.6 g/L and 31 g/L PKC412, respectively, in the Comet assay (b). At both concentrations, PKC412 led to a transcriptional downregulation of xpc, the product of which is important for DNA damage recognition. The Comet assay was performed to evaluate whether the alteration of xpc transcription has a correlate at the physiological level. PKC412 (and the positive control hydrogen peroxide) induced a dose-dependent increase in DNA damage (tail moment). Microarray data are expressed as average fold change (log2 ) with standard error compared to controls, and Comet assay values are presented as the mean SEM. *, signicantly different from controls (p < 0.05). Adapted from Oggier et al. (2011).
Box 4: General insights gained by transcriptomics. Transcriptional proles of known model compounds in sh reveal that many more genes and pathways are altered than previously thought. Transcriptomics aids in the identication of new toxicological effects as well as molecular, cellular, and biochemical pathways. It contributes to our understanding of the molecular effects of a compound. However, studies often support established knowledge, e.g. vitellogenin induction for estrogenic compounds. Expressional proles and patterns are often so complex that no clear pathways or modes of action can be identied. It is often difcult to delineate the specic molecular effects of a given compound from more general cellular stress responses. Therefore, numerous cellular and biochemical pathways reveal alterations, often based on cellular stress, apoptosis and repair mechanisms. In general the studies have shown the complex nature of a chemicals effect on transcript expression of genes at a specic point in time (usually short exposure time) and at few concentrations (usually two). Different time points and concentrations often result (though not always) in different expressional proles which complicates our understanding of the specic effects of a compound. Proles differ between tissues, making identication of a general mode of action of a compound difcult. This is also true for the delineation of the specic effects of a compound from general cellular damage and adaptive or repair mechanisms. Only a few studies have tried to link the transcriptional changes with physiological effects, or generally, toxicological effects at a higher level of biological organisation or even at population level.
waters, and is also accumulates in biota (Fent et al., 2010). EHMC was found to interact with the hormonal system of sh (Christen et al., 2011). Subsequently, we performed trancriptomics in zebrash to suggest molecular effects and to identify additional pathways affected (Zucchi et al., 2011a). EHMC lead to the alteration of as many as 1100 transcripts in whole-body analyses after 14 d of exposure. The affected genes belonged to many different pathways (lipid biosynthesis and metabolism, vitamin A metabolic process, DNA damage and apoptosis, regulation of cell growth etc.) as well as hormonal pathways. The analysis showed tissue-specic gene proles, revealing that EHMC produces a complex pattern of transcriptional changes. This study also points to a more general insight gained by transcriptomics: a compound may result in alteration of a surprisingly large number genes. The proof of concept, i.e. that unknown mechanisms of actions of a compound can be unravelled, is still yet to be provided by transcriptome studies. More compounds need to be analyzed. Only then can we judge whether or not a toxicological characterization of compounds with unknown toxicological activity can be predicted in aquatic toxicology based on the transcription (or proteome) prole. 2.2.3. Classication of compounds and new biomarkers By analyzing model compounds we also hope to discover a unique expression prole specic for compounds acting by a specic mode of action, or an expression prole of a specic tissue (e.g. brain, liver). Additionally, by using microarrays, we hope to discover new biomarkers, or expressional proles, that are typical for target tissues or modes of action. For instance, a comparison of the hepatic tumor promoters indole-3-carbinol, E2, and b-naphtoavone in rainbow trout showed that the expression prole of indole-3 carbinol resembled that of E2. However, a general feature of tumor-promoting compounds cannot (of course, as they act via different mechanisms) be deduced (Tilton et al., 2006). Thus, a classication for such acting compounds cannot be made. Lam et al. (2008) tried to distinguish different toxicants such as PAHs and estrogenic compounds based on transcriptional proles in whole zebrash microarrays in two large experiments, applying algorithms that cluster specic toxicant proles. The aim was to derive prediction models that can differentiate between compounds of the same class and those of different classes. Networks were generated from the available human homologs mapped from the discriminatory gene sets. They were able to distinguish
effect of environmental samples is evaluated, toxicogenomics may reveal the entire set of expressional changes that can be interpreted by pathway analysis. This information can be subsequently used to propose the chemicals mode of action and associated physiological effects. Hence, toxicogenomics can be used as a predictive tool for toxicological effects. The UV-lter 2-ethyl-hexyl-4-trimethoxycinnamate (EHMC) is abundant in wastewater-contaminated surface waters, bathing
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Box 5: Fingerprints and biomarkers. So far, there seems to have been more hope and promise than real scientic achievement in detecting: (a) unique expression proles for different classes of chemicals (e.g. unique ngerprints). (b) novel biomarkers of effects specic to a chemical with a known mode of action or activity. Only a few examples are documented of compounds acting via known mechanisms of action, such as the aryl hydrocarbon or estrogen receptors (e.g. Lam et al., 2008), or through established pathways, such as the hypothalamicpituitarygonadal axis (e.g., Ankley et al., 2010), which would aid in the identication of molecular biomarkers. More research is needed to identify and characterize novel biomarkers and expression proles for different classes of chemicals.
between the aryl hydrocarbon receptor and estrogen receptor agonists. Compounds with no afnity to these receptors were also identied. Some of the genes selected as biomarkers are, however, well known, including vitellogenin and Cyp1A, respectively. Others were not clearly related to known toxicological functions or have a function that is unknown, thus rendering their value open. A summary of the state of the art in compound proling and biomarker denition based on transcriptomics is given in Box 5 . 2.2.4. Compound mixtures Mainly endocrine disrupters and metals were studied for their effects when occurring in mixtures. Mixtures of differently acting compounds were studied in an attempt to identify the specic ngerprint of a compound acting by a specic, and known mode of action, within a complex mixture. In one example, trout (O. mykiss) was exposed to six different compounds with specic modes of action, including endocrine disrupters such as EE2 (estrogen), trenbolone (androgen), the ame retardant BDE-47 (putative thyroid hormone activity), benzo(a)pyrene (Cyp1a inducer and cancerogen), diquat (oxidative stress) and hexavalent chromium (oxidative stress, cancerogen) (Hook et al., 2006). The transcript expression proles of the mixture often contained many transcripts that could be related to the mode of action of a given compound. However, it is not possible to univocally identify the net effect of this mixture or the components of that mixture causing the key expression changes based on expression proles. A mixture may evoke different expressional proles, not being merely the sum of the mixture components. Vandenbrouck et al. (2009) studied binary mixtures of metals in Daphnia. The data showed additionally affected pathways after mixture treatment, indicating interactive molecular responses which are not merely the additive sum of the individual metals. These ndings indicate the complex nature of mixture toxicity on the expressional level. It seems that it is too early to assess effects of mixtures based on transcript expression proles, unless the expression proles of the individual compounds are clearly dened. And this is truly where the challenge lies. Therefore, more should be learned rst on the individual proles of single compounds. 2.2.5. Efuents and environmental samples It is even more challenging to analyze contaminated environmental samples by microarrays and to identify the compounds responsible for adverse effects by transcript expression analyses of genes. By performing microarray studies on environmental samples it is hoped that from the gene expression prole resulting from
exposure to chemical mixtures or environmental samples, specic elements can be deduced, which can be traced to the individual chemicals responsible. For example, a cross-species microarray has been used in turbot to detect the presence of a particular class of chemicals in an environmental efuent (Baker et al., 2009). Transcript expression proles of genes of unexposed and exposed sh were also compared (Fisher and Oleksiak, 2007). The data indicate that up to 17% of genes in the brain relating to metabolism displayed adaptive changes in the polluted site. In European ounder, transcriptomics and bioinformatics were used to predict the site of origin of sh from the environment (polluted or non-polluted), based on stressresponsive genes (Falciani et al., 2008). Altered expression may show the potential to identify patterns of genes associated with different types of pollution, although many environmental factors may affect the expressional pattern. In another study, wastewater efuents were analyzed by microarrays in carp (Moens et al., 2007). The data revealed that the main predicted molecular pathways affected were associated with the energy balance of the sh and digestive enzymes, but not with endocrine disruption or the immune system response. The total activity could not be assigned to the effects of specic chemicals. In another study, hepatic transcriptomics and protein analysis data showed that exposure of rainbow trout, for 14 d, to wastewater efuents affected transcript expression of stress-related genes, hormone receptor genes, and genes related to immune function (Ings et al., 2011). In addition to altered transcript expression of hepatic genes, elevated plasma cortisol, glucose, and vitellogenin levels were noted. In fathead minnows, transcript expression data were compared with expression proles of target genes in testis and liver of model compounds, EE2 and 11-ketotestosterone, as well as with vitellogenin induction and competitive nest holding behavior (Garcia-Reyero et al., 2011). Signicant changes in transcript expression were observed in both liver and testis, which correlated to vitellogenin induction and reduced competitive behavior. Microarray results showed that the transcript expression signature from efuent-exposed sh were in some part similar to estrogen and androgen signatures, which contrasted with the data by Moens et al. (2007). However, overall, they were different, revealing the complexity of compounds present in sewage and their different modes of action. Overall, not much progress seems visible based on these studies when compared to former, similar, studies performed using more classical techniques. A simulated diesel oil spill was evaluated by microarrays in conjunction with measurements of survival and growth (Mos et al., 2008). The study showed changes in genes including cyp, gluthatione S transfereases, both known biomarkers of compounds acting via AhR, as well as other genes. Additionally, new series of genes regulated by diesel were found, such as endocrine disruptive effects in the form of transcriptionally upregulated estrogen-dependent genes, and transcriptionally downregulated testosterone-related and thyroid hormone-dependent genes, as well as genes indicating immune system alterations. Gene transcription changes were found to be more sensitive than effects on growth and survival. Box 6 summarizes current state of play on the use of toxicogenomics to improve our understanding of how complex mixtures of chemicals affect sh. 2.3. Limitations and questions On the basis of current knowledge and development the following question arises: Is technical innovation also resulting in scientic innovation? The current inuence that DNA microarrays are having in medicine and biology, and also toxicology, is remarkable, but is this also true for ecotoxicology? The use of transcriptomics in the eld of ecotoxicology seems to have raised just
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Box 6: Mixtures and environmental samples. Characterization of the effects of dened mixtures of chemicals, or of ill-dened complex environmental matrices, remains extremely challenging, and the new technologies have not yet provided much additional useful information. The new technologies mainly demonstrate that exposed organisms differ from non-exposed ones; they have not provided data that can be easily interpreted and they essentially conrm what was previously known. It appears premature to tackle these issues using sophisticated and expensive technologies (transcriptomics, proteomics, metabolomics). Major insights and advances can be gained using these technologies only after a reasonable number of important selected environmental chemicals have been investigated. If unique ngerprints for different classes of chemicals can be established, these may eventually allow the responses to complex, environmentally relevant mixtures to be interpreted. There is a long way to go before mixtures and environmental samples can be assessed through meaningful studies using these technologies that will provide added value to current whole-efuent testing procedures.
It seems that there is too little hypothesis-driven research, and often little thought has gone into experimental design. In our opinion, some of the basic questions that need to be addressed have not yet been, sufciently. This conclusion strongly suggests that technical innovation is driving the use of microarrays as well as other similarly demanding techniques (proteomics, metabolomics), rather than the technology being used only where appropriate to answer specic scientic questions that are difcult, or impossible, to answer through the use of other techniques. The following list some of the critical problems surrounding this issue: (a) Technical challenges and variability of data Experiments in aquatic toxicology using transcriptomics have so far been diverse in many aspects, encompassing different microarrays (both hetero- and homologuous), organisms (sh, invertebrates, algae), exposure routes, single chemicals, mixtures and environmental samples. They represent case studies that do not follow a standardized protocol. Some validation and standardization of organisms, study design, data collection and interpretation will be necessary in the future, as variability is an ongoing concern with these technologies. There are also some rather technical issues to be solved. Currently, different signicance levels are dened, some authors consider expressional changes by 1.5 times as sufcient, although more often a factor of 2 is considered as a relevant signicance level. Inter-laboratory microarray comparisons have demonstrated that the use of non-stringent gene ltering results in non reproducible gene lists with little correlation to real-time PCR validation (Chen et al., 2007). Many studies lack appropriate bioinformatics and statistical support. Additional sources of variability, and possible technical artefacts, concerning the experimental design, such as inadequate sample numbers and chemical concentrations, methods of processing, and analysis (normalization, statistics) are common. Inter-individual variability between the responses of exposed organisms is not sufciently known, and very rarely, individuals are analyzed (normally pooled samples are analyzed). (b) Interpretation of expressional changes Even with zebrash many transcripts cannot be ascribed to a known proteins, but are assumed as having the same function as in humans. The annotation of genes is even more difcult in heterologous microarrays, where most genes have to be ascribed to genes of a related species. In general, annotation of genes in non-mammalian organisms remains a challenge. Functional annotation relies on genes with known functional characteristics. However, this is lacking for most genes in sh or lower animals or plants. Therefore, annotation is mainly based on similarity to known genes in mammals. The more distant the organism is to mammals, the fewer genes can be annotated. In addition, the extent of the variability in transcript expression data between individual organisms and repeat experiments has not been explored, and inter-laboratory analyses have not been performed, to our knowledge. Moreover, the variability of expressional changes between individual organisms is poorly characterized. We do not think that extensive and expensive inter-calibration exercises are necessary, but we think that a minimal knowledge about the accuracy of data and inter-individual variability should be gained to aid in understanding the data. Even the time of sampling and the light regime may inuence the expressional response. For instance, light induces gene transcription in zebrash, as shown by the number of 117 light-regulated genes involved in circadian rhythms, stress response, and DNA repair (Weger et al., 2011).It is often assumed that gene transcriptional proles may be similar within classes of contaminants (i.e. metals, endocrine disrupters, PAHs etc.). Yet this has not been clearly demon-
as many questions as it has answered. Among the many reasons for this is the complexity of data, its interpretation, and the lack of knowledge of the mechanisms of action of chemicals. It should be noted that gene expression proling using microarrays so far represents a snapshot of the gene transcriptional responses occurring at a given point in time, based on between one and a maximum of three concentrations, and within a particular tissue. While sequence information of genes for many aquatic species has become more accessible, their function still remains a challenge. Functional annotation identies genes with known functional characteristics, such as molecular function, pathways, or biological processes. The basic assumption of annotation is that similar genes have an identied function in other organisms. For example, the genes in zebrash are similar to those in humans. However, even zebrash has a high percentage of genes with unknown functions. The more distant the species is from mammals, the more difcult the functional annotation of the genes that are altered upon chemical exposure. Therefore, less useful information will be obtained with more distant species such as algae or mussels, unless more is known on their gene functions. There are many key open questions associated with these new technologies assessing a large number of mRNA (transcriptomics), proteins (proteomics), or small molecules (metabolomics). Out of these, most progress has been made in transcriptomics, over the last decade, whereas, to date, small progress has been made on proteomics and metabolomics. The kinetics of chemicallyinduced transcript and protein expression can be complex and dose-dependent. Core questions are still not sufciently answered such as What do these alterations really mean in terms of adverse effects? Often, alterations are identied in a largely descriptive manner and are not coupled with relevant hypotheses. How do transcript (transcriptomics) and protein (proteomics) expression, as well as metabolite (metabolomics) concentrations, relate to physiological and ecological outcomes? A link between the molecular (transcriptomics) and biochemical responses to adverse alterations in physiology, growth, development, reproduction, and survival is necessary for better understanding of adverse effects of chemicals.
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strated, although attempts have been made to pinpoint the chemicals action on the HRG axis, for instance (Wang et al., 2010a,b; Ankley et al., 2010). But it is conceivable that similar gene expression proles may eventually diverge to produce different toxicological effects. The protein kinase inhibitor PK412 affects a multitude of distinct biochemical pathways (Oggier et al., 2011). This leads to a diverse expressional pattern of many different pathways. In this and other cases, microarray data become exceedingly complex to analyze. In many cases employing whole genome microarrays, a relatively large number of genes are altered, and a multitude of transcript expression patterns are altered in response to chemical exposure (Oggier et al., 2011, Zucchi et al., 2011a). These expression patterns may in part be associated with cellular damage/repair processes. In contrast, expression proles induced by different contaminants may converge to yield similar toxicological effects, for example reduction in fertility and reproduction. Therefore, we think that it is very demanding to use toxicogenomics by clustering of expression responses to predict mechanisms of toxicity. (c) Basic toxicological concepts Concentration-response data As the concentration of a chemical increases, does the response also increase? Does the fold-increase (or decrease) in expression of a gene change in the expected manner with the dose/concentration? Are the same genes altered at different concentrations, or are more genes affected as the concentration increases? Current data show that some expressional changes are consistent between different doses, but there are also distinct differences in the transcription proles at different doses. Patterns and the reasons for these differences have not yet been fully addressed. For instance, uranium showed different expressional patterns in the brain of zebrash at two different concentrations (Lerebours et al., 2010). Time-course data How does the gene expression prole change with time? Mostly, only one or two time points are studied. However, gene expression undergoes distinct temporal phases in response to toxicants, but do phases of expression correlate with toxicological effect responses to contaminants? Temporal patterns in gene expression may provide an important representation of the regulation that occurs in response to toxic effects in time. This question needs to be addressed more deeply. The data may lead to a categorisation of different toxicity phases, in which cells/tissue rst respond to the initial exposure, followed by counter-active responses that ensure continued cellular and physiological function, and survival of the organism, during and after toxicant exposure. Data from different tissues and life stages Mostly, studies on transcript expression proles in sh are performed with embryos or the livers or the brains from adults. But if the compound is a reproductive toxicant (an estrogen, androgen, or antiandogen, for example) does liver analysis make sense? Reproductive toxicants seem to act primarily on the HPG axis. Not many studies have looked at expression proles in different tissues from the same sh, to see how similar or different they are. Targeted expression approaches reveal different tissue-specic expressional changes, as, for example, in the case of UVlters (Zucchi et al., 2011a,b). The alternative, to analyze whole sh samples, also has its disadvantage; the overall expressional prole as a sum of all changes in all tissues does not allow the identication of the target tissues. Only when the target tissue is known can this dilemma be solved. In the case of MeHg, both brain (Klaper et al., 2006) and muscle tissue (Cambier et al., 2010) were analyzed, revealing
different expressional proles, and few genes and predicted pathways were common between the tissues. Certainly, the nervous system is the target of MeHg, thus rendering analysis of brain tissue more meaningful. In case of the UV-lters benzopheone-4 and EHMC, we observed distinct expression proles in different organs (Zucchi et al., 2011a,b). Also, not many studies have compared transcript expression changes of genes in different life stages such as embryos and adult tissues. In case of the pharmaceutical diazepam, we found a good correlation between different life stages (Oggier et al., 2010), but more research has to be devoted to compare tissues and life stages. We think much more of the baseline data are required in order to be able to meaningfully interpret the results of a microarray exposure experiment. An example is the comprehensive list of developmentally regulated zebrash genes and their transcript expression proles during embryogenesis provided by Mathavan et al. (2005) including novel information on the temporal expression of several thousand previously uncharacterized genes. (d) Costs, training, bioinformatics capabilities Toxicogenomics is a demanding technology that is costly and requires great skill. In addition, individual responses (variability), data processing, and complexity of data interpretation remain obstacles. (e) Ecotoxicological relevance Toxicogenomics has shown its value in identifying new modes of toxic action of contaminants that were previously unexpected. However, the link between transcript expression changes and effects at higher biological levels has rarely been addressed. In particular, integrative assessments of the changes in gene transcription and physiology encoding for adverse health effects are scarce. To date, transcriptomics has rarely been assessed in concert with physiological, histological and phenotypic measures of chemical exposure. Thus, often trancriptomic data remain descriptive, though they can provide insights into new pathways affected by the compound. Whether or not these changes translate into physiological adverse effects remains in many cases unknown. Forthcoming studies must therefore try to establish solid links between expressional changes and responses measured at the cell or tissue level and adverse outcomes traditionally measured at the individual or population level.
3. Proteomics and metabolomics We mentioned that expression signatures often lack any relation to the responses of the organism, such as physiological changes, growth, fertility or reproduction, thus limiting their value. Focusing on the transcription of genes is only an intermediate step in the process of converting genetic information into proteins and physiological function. Ideally, the information on gene transcription should be complemented by information on the protein, metabolic, and physiological levels. Similar to toxicogenomics, alterations in protein proles (proteomics) or in the metabolites prole (metabolomics) have developed as novel techniques to determine effects of chemical exposure. They also lend themselves to generating an enormous amount of information. To date, advances in aquatic toxicology have mainly been achieved in toxicogenomics, whereas proteomics and metabolomics are rarely applied. Changes in the mRNA level do not necessarily translate to the protein or physiological level. Proteomics determines the occurrence of a series of proteins in a cell, organ or in body uids, whereas metabolomics determines the occurrence of generally
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small molecules in body uids or tissues. Both techniques are also challenging and need sophisticated analytical chemical equipment, for proteomics 2D electrophoresis coupled with MALDI-TOF MS and MS/MS, and for metabolomics, nuclear magnetic resonance spectroscopy. So far, only a very few chemicals have been analyzed by proteomics. This is probably due to the complexity, and lack of functional protein annotation and costs. Metabolomics seems to have more applications. However, similar to trancriptomics, proteomic and metabolomic techniques are very expensive and demanding. The challenges and questions associated with these techniques are similar to those with transcriptomics. 3.1. Proteomics The assessment of a series of tens to hundreds or even thousands of proteins in tissues or body uids, combined with functional annotation and pathway analysis, offers information about cellular functions pathways and processes involved. One important aspect in toxicogenomics is to determine how transcript expression is correlated to proteins. Currently, it is poorly understood how well an increase in mRNA correlates with increased protein abundance. The half-lives of mRNA and protein often differ; therefore, protein proles are not necessarily identical to the mRNA expression proles. Studies analyzing both proles in combination are very rare. To date there are only a few quantitative proteomic-based studies in sh, and less in invertebrates such as Daphnia (Frhlich et al., 2009). In rainbow trout liver, 15 proteins were altered after wastewater exposure (Albertsson et al., 2007). Also proteomic response to hypoxia has been studied in medaka (Oehlers et al., 2007). Proteomics has been used to evaluate the effects of mycrocystin-LR (Wang et al., 2010a,b), peruorooctane sulfonate (Shi et al., 2009), ame retardants (De Wit et al., 2008; Kling and Frlin, 2009), trenbolone (Martyniuk et al., 2009), cadmium (Zhu et al., 2006; Ling et al., 2009), and wastewater (Albertsson et al., 2007). Usually, only one or very few concentrations were analyzed, and only at one point in time. The studies also show that many altered proteins could not be identied, and only a small set of proteins were actually identied and interpreted. Exposure of zebrash to microcystin-LR caused damage to liver cellular ultrastructure, and the abundance of 22 proteins was remarkably altered in response to toxin exposure (Wang et al., 2010a,b). Among them 19 proteins could be identied and annotated to cytoskeleton assembly, macromolecule metabolism, oxidative stress, and signal transduction. This study shows that protein proles correlate with ultrastructural changes, and indicates additional effects (oxidative stress) to those previously known. Responses of MeHg in the brain of Atlantic cod (Gadus morhua) revealed 71 protein spots, of which 40 were identied, to be significantly altered following injection of the metal into the body (Berg et al., 2010). Many of the proteins were associated with known targets and mechanisms of MeHg, while several of them have not previously been associated with MeHg exposure. Kling and Frlin (2009) analyzed the proteome response of zebrash liver cells after 24- and 72-h exposure to hexabromocyclododecane (HBCD), TBBPA, as well as a mixture of both. A total of 7 altered proteins in response to HBCD, two in response to TBBPA, and 9 in response to the mixture, were identied. Zebrash embryos exposed to 0.5 mg/L peruoroocane sulfonate (PFOS) at a single point in time (192 hpf) expressed 69 altered proteins, 38 of which were analyzed and 18 proteins identied. These proteins can be annotated to different functional classes such as detoxication, energy metabolism, lipid transport/steroid metabolic process, cell structure, signal transduction, and apoptosis. However, the other altered proteins remain unknown and their functional roles remain yet to be identied, thus revealing the limitations to the insight that can be gained from proteomic analysis.
3.2. Metabolomics As with transcriptomics and proteomics, many fundamental requirements must be met so that metabolomics can be applied in toxicological assessment of environmental chemicals. Similar to the other techniques, metabolomics also represents a snapshot in time, in this case of the low molecular weight metabolites that can change upon exposure to environmental chemicals. Whether metabolomics may ultimately serve as a diagnostic tool or a method for a better understanding of a chemicals adverse effects remains unknown. It will be a long time before we can use metabolomics for analysis of biota in contaminated environments or in any sort of risk assessment. An inter-comparison exercise with seven laboratories resulted in comparable results in liver extracts of European ounder, and the same set of metabolites differentiated sh from clean sites from those from contaminated sites (Viant et al., 2009). Urinary metabolite proles were assessed in fathead minnows after exposure to model compounds, which in the case of cyproterone acetate was not very different from that of controls, but differed after coexposure to the androgen trenbolone (Collette et al., 2010). A targeted metabolite proling performed in roach (Rutilus rutilus) exposed to EE2 in bile and plasma, as well as in liver and gonads, showed that sex steroid and glucocorticoid pathways were altered at 10 ng/L (Flores-Valverde et al., 2010). Metabolic changes were assessed in earthworms after exposure to 3-uoro-4-nitropheol (Bundy et al., 2001) or pyrene (Jones et al., 2008). Both site- and contaminant-specic effects on the metabolic proles could be discerned from earthworms from seven sites with different levels of metal contamination (Bundy et al., 2007). Metabolic signatures have also been assessed in chemically exposed marine mussels (Mytilus edulis) to two copper and pentachlorophenol concentrations for 7 d, and subsequently, mussels were collected in the eld from rural and industrialized sites and their metabolome compared to that of laboratory-exposed mussels (Hines et al., 2010). Both compounds induced metabolic and physiological changes, and the metabolic signatures predicted a reduced tness of mussels from contaminated sites. Box 7 summarizes the current state of these technologies.
4. Combination of toxicogenomics, proteomics and metabolomics More research is needed to relate transcriptional changes with effects on the physiology of the organism or with generally assessed parameters of toxicity. Often, changes in transcripts, proteins or metabolites are purely descriptive, without links to known physiological effects of toxicants. Additionally, only a very few studies have been performed trying to combine toxicogenomics with proteomics and/or metabolomics. Aquatic toxicology should move towards the integration of genomic data (and possibly proteomic, and metabolomic data) to better understand the underlying physiology and toxicology rather than being purely descriptive (e.g. which genes are affected at what concentrations). Bioinformatics approaches, such as a pathway analysis, will be important in providing functional insight into genomics and physiological outcomes. Studies integrating both transcriptome and proteome analyses are rare. De Wit et al. (2008) analyzed the transcriptional changes in the liver of adult zebrash exposed to two concentrations of tetrabromobisphenol-A (TBBPA), as well as one concentration of TBBP, for protein alterations. The data indicate that TBBPA interferes with the thyroid and vitamin A homeostasis, affects the energy balance, the onset of oxidative stress, and causes a general stress response. Additionally, numerous differentially expressed tran-
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Box 7: State of the art of proteomics and metabolomics. It is necessary for a sequenced genome of a species of interest to be available for insightful proteomic analysis, but this holds true for only a few species. Great challenges have to be overcome, such as the annotation of proteins to elucidate their function. Proteomic studies have focused mostly on a single point in time, at one or two exposure concentrations. These only allow a restricted insight into the toxicological effects or modes of action. The potential of proteomics to be used to discover new mechanisms of physiological and toxic action as well as the discovery of biomarkers of exposure and effects has not yet been met. Moreover, the protein proles have rarely been linked to transcriptomics or to phenotypic or physiological effects, and their combination has not yet resulted in the discovery of signicant insights. Interpretation and combination of proteomic data with physiology and ecotoxicologically relevant measures is still lacking. Similar to proteomics, metabolomics seems to be in its infancy and currently demonstrates the proof of principle, rather than advancing knowledge of the modes of action of chemicals and/or on their adverse effects. Proteomic and metabolomic studies often conrm that exposed animals have a different protein and metabolic pattern, respectively, than unexposed ones. However, they are so far not very helpful in further elucidating effects of environmental pollutants. Whether a chemicals mode of action can be predicted by the proteomic or metabolic prole, is still open. Currently, it seems that these technically demanding methods and expensive technology has not resulted in much novel scientic insight; instead, their application has mainly conrmed what was already known.
edge. However, also some additional targets (i.e. downregulation of cholestrol biosynthesis) were noted. Some of the changes in metabolites could be related to transcriptional changes, but the potentially new insights from a combination of transcription and metabolites proles were not obvious.
5. Conclusions and outlook: where to go? The presence of chemicals that cause adverse effects on wildlife and/or humans is of great concern. The identication of chemicals of most concern (out of the very large number present) is important. We want to know what effects they cause, either individually or in mixtures, and what degree of environmental risk is posed by those effects. We are convinced that the new technologies, in particular the microarray technology, can help to address, and hopefully answer, some of these questions. As environmental scientists our aim is to protect the environment from the threats it faces. Thereby, these novel technologies could, in the future, play a very helpful role in environmental risk assessments of toxicants, but we think that they need to be used more wisely than they currently are. Toxicogenomics has important merits in elucidating the modes of action of chemicals and reecting their molecular effects. This technology allows a broader view of possible mechanisms by which compounds act, and even gives the entire picture of its effects. However, these merits can only be met when baseline data are known, and a proper experimental design is used. Toxicological concepts, including proper doseresponse curves, choice of target tissues (preferably multiple tissues), and time-courses, should be investigated. Currently, toxicogenomics, and certainly proteomics and metabolomics, are in a research state. Data of these technologies are not yet fully understood, and therefore cannot be meaningfully utilized for ecological risk assessment of environmental toxicants unless these technologies are combined and associated with toxicological and physiological measurements. Gene expression proling, using microarrays, and also proteomics and metabolomics, are new tools in aquatic toxicology. They are complex and sophisticated tools, and very expensive. So far, the vast majority of published studies provide descriptions only of how genes, or proteins, or metabolites are altered by stresses. Some studies attempt to link those changes with physiological processes. However, research should be driven by scientic questions and hypotheses, not the availability of (and fascination for) a new technique. The question should dictate the study, and what experimental techniques are used in that study, whereas presently much of the research appears to originate rather from the fascination for a new and sophisticated tool. We should not forget that every technique has advantages as well as its limitations. It seems that we are moving too fast in this eld and want to answer big questions before we have answered the basic ones. The fascination for a sophisticated and demanding tool seems to hinder the posing of critical questions that aid in the advancement of science. Better baseline data are needed in order that effects (changes to the baseline) can be identied and interpreted correctly. Therefore, studies should be conducted to dene baseline activities (gene expression, protein or metabolic proles). Without such data a huge amount of time and resources will be wasted. It is too premature to analyze mixture effects or environmental samples by means of transcriptomics, proteomics or metabolomics. First, proles of single compounds, their reproducibility, their value and potential for making predictions (such as utility as biomakers) have to be demonstrated before more complex matrices can be investigated for meaningful insights. In forthcoming studies more emphasis should be placed on understanding the expressional, protein, or metabolite proles
scripts could be associated with cellular defence mechanisms and cellular metabolism. The study also shows that many of the altered hepatic proteins could not be identied. To a small extent, the observed responses at the protein level were conrmative of the mRNA changes, but a correlation between transcriptional and protein proles was demonstrated for only a few proteins (e.g. stress reaction, gluconeogenesis). Thus, both responses seem to demonstrate rather their unique proles, rather than being correlated and conrmative; they seem rather complementary. The potential of a combined genomics and proteomics approach to elucidate molecular effects has yet to be demonstrated. In stickleback (Gasterosteus aculeatus) a known cancerogenic PAH, 1,2:5,6-dibenzanthracene, was studied for its effects on the transcription of genes in the liver and on the metabolite prole at three concentrations after 4 d of exposure (Williams et al., 2009). Induction of expression of known genes (cyp) and genes related to bile acid biosynthesis, steroid metabolism and endocrine function were found, while metabolomics revealed notable changes in taurine, malonate, glutamate and alanine. This study showed some relationships between transcriptional changes and metabolic responses. Also copper (Cu) was studied in stickleback using transcriptomics and metabolomics in the liver after exposure to 3.2128 g/L for 4 d (Santos et al., 2010). Cu also resulted in increased DNA damage in blood cells. Transcriptional effects were in line with previously documented effects (i.e. induction of oxidative stress). Metabolomics data supported known mitochondrial effects (i.e. effect on oxidative phosphorylation). The effects noted include those that were expected on the basis of previous knowl-
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K. Fent, J.P. Sumpter / Aquatic Toxicology 105S (2011) 2539 Collette, T.W., Teng, Q., Jensen, K.M., Kahl, M.D., Makynen, E.A., Durhan, E.J., Vil leneuve, D.L., Martinovic-Weigelt, D., Ankley, G.T., Ekman, D.R., 2010. Impacts of an anti-androgen and an androgen/anti-androgen mixture on the metabolite prole of male fathead minnow urine. Environ. Sci. Technol. 44, 68816886. Connon, R., Hooper, H.L., Sibly, R.M., Lim, F.-L., Heckmann, L.-H., Moore, D.J., Watanabe, H., Soetaert, A., Cook, K., Maund, S.J., Hutchinson, T.H., Moggs, J., De Coen, W., Iguchi, T., Callaghan, A., 2008. Linking molecular and population stress responses in Daphnia magna exposed to cadmium. Environ. Sci. Technol. 412, 21812188. Copeland, P.A., Sumpter, J.P., Walker, T.K., Croft, M., 1986. Vitellogenin levels in male and female rainbow trout Salmo gairdneri Richardson at various stages of the reproductive cycle. Comp. Biochem. Physiol. B 83, 487493. De Wit, M., Keil, D., Remmerie, N., van der Ven, K., van den Brandhof, E.J., Knapen, D., Witters, E., De Coen, W., 2008. Molecular targets of TBBPA in zebrash analysed through integration of genomic and proteomic approaches. Chemosphere 74, 96105. Diab, A.M., Williams, T.D., Sabine, V.S., Chipman, J.K., George, S.G., 2008. The GENIPOL European ounder Platichthys esus L. toxicogenomics microarray: application for investigation of the response to furunculosis vaccination. J. Fish Biol. 72, 21542169. Falciani, F., Diab, A.M., Sabine, V., Williams, T.D., Ortega, F., George, S.G., 2008. Hepatic transcriptomic proles of European ounder (Platichthys esus) from eld sites and computational approaches to predict site stress gene responses following exposure to model toxicants. Aquat. Toxicol. 90, 92101. Fent, K., Zenker, A., Rapp, M., 2010. Widespread occurrence of estrogenic UV-lters in aquatic ecosystems in Switzerland. Environ. Pollut. 158, 18171824. Filby, A.L., Thorpe, K.L., Maack, G., Tyler, C.R., 2007. Gene expression proles revealing the mechanisms of anti-androgen-and estrogen-induced feminisation in sh. Aquat. Toxicol. 81, 219231. Fisher, M.A., Oleksiak, M.F., 2007. Convergence and divergence in gene expression among natural populations exposed to pollution. BMC Genomics 8, 108. Flores-Valverde, A.M., Horwood, J., Hill, E.M., 2010. Disruption of the steroid metabolome in sh caused by exposure to the environmental estrogen 17 ethinylestradiol. Environ. Sci. Technol. 44, 35523558. Frhlich, T., Arnold, G.J., Fritsch, R., Mayr, T., Laforsch, C., 2009. LC-MS/MS-based proteome proling in Daphnia pulex and Daphnia longicephala: the Daphnia pulex genome database as a key for high throughput proteomics in Daphnia. BMC Genomics 10, 171. Garcia-Reyero, N., Barber, D.S., Gross, T.S., Johnson, K.G., Sepulveda, M.S., Szabo, N.J., Denslow, N.D., 2006. Dietary exposure of largemouth bass to OCPs changes expression of genes important for reproduction. Aquat. Toxicol. 78, 358369. Garcia-Reyero, N., Adelman, I., Liu, L., Denslow, N., 2008. Gene expression proles of fathead minnows exposed to surface waters above and below a sewage treatment plant in Minnesota. Mar. Environ. Res. 66, 134136. Garcia-Reyero, N., Villeneuve, D.L., Kroll, K.J., Liu, L., Orlando, E.F., Watanabe, K.H., Sepulveda, M.S., Ankley, G.T., Denslow, N.D., 2009. Expression signature for a model androgen and antiandrogen in the fathead minnow (Pimephales promelas) ovary. Environ. Sci. Technol. 43, 26142619. Garcia-Reyero, N., Lavelle, C.M., Escalon, B.L., Martinovic, D., Kroll, K.J., Sorensen, P.W., Denslow, N.D., 2011. Behavioural and genomic impacts of a wastewater efuent on the fathead minnow. Aquat. Toxicol. 101, 3848. Griftt, R.J., Weil, R., Hyndman, K.A., Denslow, N.D., Powers, K., Taylor, D., Barber, D.S., 2007. Exposure to copper nanoparticles causes gill injury and acute lethality in zebrash (Danio rerio). Environ. Sci. Technol. 41, 81788186. Gunnarsson, L., Kristiansson, E., Frlin, L., Nerman, O., Larsson, D.G.J., 2007. Sensitive and robust gene expression changes in sh exposed to estrogena microarray approach. BMC Genomics 8, 149. Hines, A., Staff, F.J., Widdows, J., Compton, R.M., Falciani, F., Viant, M.R., 2010. Discovery of metabolic signatures for predicting whole organism toxicology. Toxicol. Sci. 115, 369378. Hoffmann, J.L., Torontali, A.S.P., Thomason, R.G., Lee, D.M., Brill, J.L., Price, B.B., Carr, G.J., Versteeg, D.J., 2006. Hepatic gene expression proling using GeneChips in zebrash exposed to 17 -ethynylestradiol. Aquat. Toxicol. 79, 233246. Hoffmann, Z.J., Thomason, R.G., Lee, D.M., Brill, J.L., Price, B.B., Carr, G.J., Versteeg, D.J., 2008. Hepatic gene expression proling using GeneChips in zebrash exposed to 17 -methyldihydrotestosterone. Aquat. Toxicol. 87, 6980. Hogstrand, C., Balesaria, S., Glover, C.N., 2002. Application of genomics and proteomics for study of the integrated response to zinc exposure in a model sh species, the rainbow trout. Comp. Biochem. Physiol. B 133, 523535. Hook, S.E., Skillman, A.D., Small, J.A., Schultz, I.R., 2006. Gene expression patterns in rainbow trout, Oncorhynchus mykiss, exposed to a suit of model toxicants. Aquat. Toxicol. 77, 372385. Ings, J.S., Servos, M.R., Vijayan, M.M., 2011. Hepatic transcriptomics and protein expression in rainbow trout exposed to municipal wastewater efuent. Environ. Sci. Technol. 45, 23682376. Jones, O.A.H., Spurgeon, D.J., Svendsen, O., Grifn, J.L., 2008. A metabolomics-based approach to assessing the toxicity of the polyaromatic hydrocarbon pyrene to the earthworm Lumbricus rubellus. Chemosphere 71, 601609. Kausch, U., Alberti, M., Haindl, S., Budczies, J., Hock, B., 2008. Biomarkers for exposure to estrogenic compounds: gene expression analysis in zebrash (Danio rerio). Environ. 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with respect to the toxicants action, instead of on mere description of how many genes, proteins or metabolites are altered and what processes may be affected. We are convinced that transcriptomics, proteomics, and metabolomics must be related to physiology and toxicological effects at the tissue or organism level. This is the direction we should go in the future. Without linking the many changes measured to physiological and toxicological outcomes (e.g. functional analyses), they remain descriptive and do not advance our knowledge of potential effects of compounds. So far, in most studies it remains unclear whether or not measured changes in transcript expression, protein, or metabolic proles should be considered adverse. Whether or not these changes translate to physiological changes, and whether those, in turn, lead to reduced (ecological) tness (in particular, development, reproduction, survival) is the core point. Studies relating such changes to physiology are needed in the future to make benecial use of the new technologies and advance ecotoxicology. A nal point: we wrote this article from an ecotoxicological perspective, as we are interested in the effects of chemicals on wildlife to help preventing hazards. But we should be aware that these new technologies, especially transcriptomics, demonstrated their extreme usefulness in medicine and biology, and to some extent in toxicology. Any environmental change will, presumably, alter gene expression patterns, as biota respond to that change and adapt. For example, transcriptomics could be a useful tool in investigating how aquatic organisms will respond to environmental changes, including climate change. This will show what physiological mechanisms are involved in adaptation to these environmental changes. References
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Aquatic Toxicology 105S (2011) 2539

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K. Fent, J.P. Sumpter / Aquatic Toxicology 105S (2011) 2539

K. Fent, J.P. Sumpter / Aquatic Toxicology 105S (2011) 2539

K. Fent, J.P. Sumpter / Aquatic Toxicology 105S (2011) 2539

K. Fent, J.P. Sumpter / Aquatic Toxicology 105S (2011) 2539

K. Fent, J.P. Sumpter / Aquatic Toxicology 105S (2011) 2539

K. Fent, J.P. Sumpter / Aquatic Toxicology 105S (2011) 2539

K. Fent, J.P. Sumpter / Aquatic Toxicology 105S (2011) 2539

K. Fent, J.P. Sumpter / Aquatic Toxicology 105S (2011) 2539

K. Fent, J.P. Sumpter / Aquatic Toxicology 105S (2011) 2539

K. Fent, J.P. Sumpter / Aquatic Toxicology 105S (2011) 2539

K. Fent, J.P. Sumpter / Aquatic Toxicology 105S (2011) 2539

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