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Genes and environment—Striking the fine balance between sophisticated biomonitoring and true functional environmental genomics
Abstract
This article provides an overview how the application of the gene profiling (mainly via microarray technology) can be used in different organisms to address issues of environmental importance. Only recently, environmental sciences, including ecotoxicology, and molecular biology have started to mutually fertilize each other. This conceptual blend has enabled the identification of the interaction between molecular events and whole animal and population responses. Likewise, striking the fine balance between biomonitoring and functional environmental genomics will allow legislative and administrative measures to be based on a more robust platform. The application of DNA microarrays to ecotoxicogenomics links ecotoxicological effects of exposure with expression profiles of several thousand genes. The gene expression profiles are altered during toxicity, as either a direct or indirect result of toxicant exposure and the comparison of numerous specific expression profiles facilitates the differentiation between intoxication and true responses to environmental stressors. Furthermore, the application of microarrays provides the means to identify complex pathways and strategies that an exposed organism applies in response to environmental stressors. This review will present evidence that the widespread phenomenon of hormesis has a genetic basis that goes beyond an adaptive response. Some more practical advantages emerge: the toxicological assessment of complex mixtures, such as effluents or sediments, as well as drugs seems feasible, especially when classical ecotoxicological tests have failed. The review of available information demonstrates the advantages of microarray application to environmental issues spanning from bacteria, over algae and spermatophytes, to invertebrates (nematode Caenorhabditis elegans, crustacea Daphnia spp., earthworms), and various fish species. Microarrays have also highlighted why populations of a given species respond differently to similar contaminations. Furthermore, this review points at inherent limits of microarrays which may not yet have been properly addressed, namely epigenetics, which may explain heritable variation observed in natural population that cannot be explained by differences in the DNA sequence. Finally, the review will address promising future molecular biological developments which may supersede the microarray technique.
... Some of these symptoms are quite general and unspecific, regardless of the nature of the stressor, such as the development of oxidative stress and induction of repairing proteins (the so-called heat shock proteins, HPSs) (Steinberg, 2011). This gave rise to the use of many enzymes and proteins, and ultimately the expression of the genes coding for them, as stress biomarkers (Steinberg et al., 2008b). The development of the "-omic" approaches-i.e., the mapping of whole-proteomic, -genomic and -metabolomic expression in organisms-led to sensitive and comprehensive methods for evaluating the effects of stress in organisms (Bijlsma & Loeschcke, 2005;Steinberg et al., 2008b). ...
... This gave rise to the use of many enzymes and proteins, and ultimately the expression of the genes coding for them, as stress biomarkers (Steinberg et al., 2008b). The development of the "-omic" approaches-i.e., the mapping of whole-proteomic, -genomic and -metabolomic expression in organisms-led to sensitive and comprehensive methods for evaluating the effects of stress in organisms (Bijlsma & Loeschcke, 2005;Steinberg et al., 2008b). ...
... The advance of genomic techniques brought up new methods for multivariate gene-expression analysis (DNA microarrays and mRNA sequencing) encompassing virtually thousands of genes. With these techniques, one may observe simultaneously which genes are up-and down-regulated when organisms are submitted to stress (Steinberg et al., 2008b). This approach has been used with cladocerans mostly from an ecotoxicological perspective-the so called ecotoxicogenomics-i.e., testing the effects of chemicals on the overall gene activity profile of organisms exposed to toxicants (Vandenbrouck et al., 2011). ...
Cladocerans are microcrustaceans component of the zooplankton in a wide array of aquatic ecosystems. These organisms, in particular the genus Daphnia, have been widely used model organisms in studies ranging from biomedical sciences to ecology. Here, we present an overview of the contribution of studies with cladocerans to understanding the consequences at different levels of biological organization of stress induced by environmental factors. We discuss how some characteristics of cladocerans (e.g., small body size, short life cycles, cyclic parthenogenesis) make them convenient models for such studies, with a particular comparison with other major zooplanktonic taxa. Then we illustrate the contribution of cladocerans to stress research with examples encompassing stress responses spanning from the molecular to the populational level. Most worth of note are recent studies that presented evidence of beneficial consequences of mild stress caused by natural stressors (cross-tolerance), which may be passed along across generations, favoring individual survival and species persistence in fluctuating environments. This would be particularly relevant for environments prone to frequent natural environmental fluctuations, such as coastal lagoons and other shallow aquatic ecosystems. Based on reviewed studies, a conceptual model is presented summarizing the potential effects of a first stressor on the organism’s resistance to a second one. We finish by highlighting some gaps on environmental stress research that could benefit from further studies using cladocerans as model organisms.
... Caenorhabditis elegans is a non-parasitic nematode which lives within the interstitial water in the soil. Characterised by a fast generation time and large brood size (Riddle et al. 1997) and its completely sequenced and fully annotated genome, C. elegans has been subjected to many genetical and biochemical studies (Steinberg et al. 2008). Some 60-80% of genes within the worm genome are orthologous to their human counterparts (Lai et al. 2000;Kaletta and Hengartner 2006), for example 12 out of 17 signal transduction pathways are conserved between humans and C. elegans (NRC 2000). ...
... More importantly, 40% of human disease-associated genes are represented by orthologs in the C. elegans genome (Culetto 2000). C. elegans has been used in the discovery of pharmacological targets for human diseases and is gaining attention as a promising multicellular alternative for studying environmental pollutants at the molecular level as well as the level of whole organisms (Meier et al. 2014;Polak et al. 2014;Steinberg et al. 2008;Volkova et al. 2020). Previous toxicological research with C. elegans mainly focused on inorganic substances, such as heavy metals (Williams and Dusenbery 1990;Harada et al. 2007) or pesticides (Jones et al. 1996;Rajini et al. 2008). ...
Benzo[ a ]pyrene (BaP) is bioactivated in most organisms by the cytochrome P450 (CYP) enzymes, mainly CYP1A1, ultimately resulting in the reactive metabolite BaP-7,8-dihydrodiol-9,10-epoxide (BPDE) capable of covalently binding to DNA and forming adducts. This step has been defined as the key process in cancer initiation in humans. However, limited knowledge is available about the consequences of BaP exposure in organisms lacking this classical CYP1A1 pathway, one example is the model nematode Caenorhabditis elegans. The aim of this study was to define the genotoxic potential of BaP in C. elegans and to advance our understanding of xenobiotic processing in the absence of the CYP1A1 pathway. Exposure to high concentrations of BaP (0–40 µM) significantly affected life cycle endpoints of C. elegans , which were manifested by a reduced reproductive output and shortened life span. An optimised comet assay revealed that DNA damage increased in a dose-dependent manner; however, no bulky DNA adducts (dG- N 2 -BPDE) were observed by ³² P-postlabelling. Global transcriptomic analysis by RNA-Seq identified responsive transcript families, most prominently members of the cyp-35 and UDP-glucuronosyltransferases (UGTs) enzyme families, both of which are linked to xenobiotic metabolism. Strains harbouring mutations in the cyp-35A2 and cyp-35A3 genes were notably less prone to BaP-mediated toxicity, and BaP led to longevity in cyp-35A5 mutants. In summary, BaP induces transcriptional, genotoxic and phenotypic responses in C. elegans , despite the absence of the classical CYP1A1 bioactivation pathway. This provides first evidence that parallel pathways are implicated in BaP metabolism in C. elegans and this seems to be mediated via the cyp-35 pathway.
... Changes in gene expression reflect rapid and sensitive responses of organisms to environmental insults. Toxicogenomics using DNA microarray platforms for ecologically relevant eukaryotes has been an auspicious tool in predictive (eco)toxicology, disclosing molecular biomarkers of toxicity and xenobiotic MoA in biological systems (reviewed in Steinberg et al. 2008). A number of gene expression profiling studies have used the yeast S. cerevisiae, revealing toxicity mechanisms of bioavailable xenobiotics, drugs and industrial effluents (reviewed in Braconi et al. 2016;Santos and Sá-Correia 2015;Yasokawa and Iwahashi 2010) and disclosing molecular indicators of xenobiotic toxicity to be used in environmental biomonitoring (Gil et al. 2015;Kim et al. 2006). ...
... To ensure comparable pesticide-triggered transcriptional responses and correlating with yeast outcomes at higher level of biological organization (growth inhibition), the genomewide transcriptional profiles were obtained in a standardized yeast population upon 2 h of exposure to concentrations of each pesticide provoking equivalent toxic effects, namely pesticide concentrations inhibiting yeast growth by 20% relative to the growth of the untreated control cells (hereafter designated as the 20%-effective concentration IC 20 ). These short-term exposure conditions associated with an impairment of low magnitude in yeast physiology were chosen in order to unveil potential early-alarm indicators of pesticide aggression before signs of eventual deterioration of cellular function (Steinberg et al. 2008). ...
Accidental spills and misuse of pesticides may lead to current and/or legacy environmental contamination and may pose concerns regarding possible risks towards non-target microbes and higher eukaryotes in ecosystems. The present study was aimed at comparing transcriptomic responses to effects of sub-lethal levels of six environmentally relevant pesticide active substances in the Saccharomyces cerevisiae eukaryotic model. The insecticide carbofuran, the fungicide pyrimethanil and the herbicides alachlor, S-metolachlor, diuron and methyl(4-chloro-2-methylphenoxy)acetate were studied. Some are currently used agricultural pesticides, while others are under restricted utilization or banned in Europe and/or North America albeit being used in other geographical locations. In the present work transcriptional profiles representing genome-wide responses in a standardized yeast population upon 2 h of exposure to concentrations of each compound exerting equivalent toxic effects, i.e., inhibition of growth by 20% relative to the untreated control cells, were examined. Hierarchical clustering and Venn analyses of the datasets of differentially expressed genes pointed out transcriptional patterns distinguishable between the six active substances. Functional enrichment analyses allowed predicting mechanisms of pesticide toxicity and response to pesticide stress in the yeast model. In general, variations in transcript numbers of selected genes assessed by Real-Time quantitative reverse transcription polymerase chain reaction confirmed microarray data and correlated well with growth inhibitory effects. A possible biological relevance of mechanistic predictions arising from these comparative transcriptomic analyses is discussed in the context of better understanding potential modes of action and adverse side-effects of pesticides.
... The invertebrate nematode Caenorhabditis elegans (C. elegans) is a useful model organism for studying toxicogenomic responses to environmental pollutants at the molecular level as well as at the level of the organism (Polak et al., 2014;Steinberg et al., 2008). Due to the availability of the whole genome sequence, C. elegans has been subjected to gene expression studies (Steinberg et al., 2008). ...
... elegans) is a useful model organism for studying toxicogenomic responses to environmental pollutants at the molecular level as well as at the level of the organism (Polak et al., 2014;Steinberg et al., 2008). Due to the availability of the whole genome sequence, C. elegans has been subjected to gene expression studies (Steinberg et al., 2008). Further, C. elegans has been shown to be a useful alternative to mammalian models because mutant strains can be generated in which distinct genes are knocked out or are genetically modified (e.g. ...
... The invertebrate nematode Caenorhabditis elegans (C. elegans) is a useful model organism for studying toxicogenomic responses to environmental pollutants at the molecular level as well as at the level of the organism (Polak et al., 2014;Steinberg et al., 2008). Due to the availability of the whole genome sequence, C. elegans has been subjected to gene expression studies (Steinberg et al., 2008). ...
... elegans) is a useful model organism for studying toxicogenomic responses to environmental pollutants at the molecular level as well as at the level of the organism (Polak et al., 2014;Steinberg et al., 2008). Due to the availability of the whole genome sequence, C. elegans has been subjected to gene expression studies (Steinberg et al., 2008). Further, C. elegans has been shown to be a useful alternative to mammalian models because mutant strains can be generated in which distinct genes are knocked out or are genetically modified (e.g. ...
This study aimed to establish a protocol for cell dissociation from the nematode Caenorhabditis elegans (C. elegans) to assess the genotoxicity of the environmental pollutant benzo[a]pyrene (BaP) using the alkaline version of the single cell electrophoresis assay (comet assay). BaP genotoxicity was assessed in C. elegans (wild-type [WT]; N2, Bristol) after 48h exposure (0–40μM). Induction of comets by BaP was concentration-dependent up to 20μM; comet% tail DNA was ∼30% at 20μM BaP and ∼10% in controls. Similarly, BaP-induced DNA damage was evaluated in C. elegans mutant strains deficient in DNA repair. In xpa-1 and apn-1 mutants BaP-induced comet formation was diminished to WT background levels suggesting that the damage formed by BaP that is detected in the comet assay is not recognised in cells deficient in nucleotide and base excision repair, respectively. In summary, our study provides a protocol to evaluate DNA damage of environmental pollutants in whole nematodes using the comet assay.
... As is the case for other animal groups, the vast majority of the toxicology and ecotoxicology literature on copepods reports on whole-animal assays, which depend on estimates of lethality or whole organism reproductive / developmental effects to assess the impact of aquatic toxicants. The last several years has seen the widespread application of technologies such as microarrays to the study of aspects of ecotoxicology (ecotoxicogenomics), environmental stress and overall ecosystem health using a wide variety of animals (reviewed in Steinberg et al. 2008). With respect to copepods molecular methods have only recently been applied in these areas for Calanus finmarchicus (Hansen et al. 2007;Hansen et al. 2009) and Tigriopus japonicus (Seo et al. 2006a,b;Lee et al. 2006;Lee et al. 2008;Rhee et al. 2008). ...
... Use of microarrays in toxicology and ecotoxicology has revolutionized these areas of research by allowing for the identification of pathways and strategies employed by organisms in response to environmental stressors (reviewed in Steinberg et al. 2008). Genomics resources would therefore assist the identification of suites of genes associated with particular physiological stressors e.g. ...
The copepods outnumber, in terms of individual animals, every other group of
multicellular animals on earth, including insects and nematode worms, and are critical
components of the world’s freshwater and marine ecosystems (Hardy 1970). Copepods
are also vectors of human diseases such as cholera and can be major pathogens of wild
and farmed aquatic animals. Despite their central importance, there are currently no
large-scale genomics resources for copepods and relatively few publically available
sequences. Here, we explain how such resources would facilitate the development of
fundamental biological knowledge, addressing key global problems and answering
questions of broad interest. This paper is a collaborative effort by members of the
international copepod research community. Its objective is to provide impetus for a
much-needed initiative to develop large-scale genomics resources for copepods.
The conference workshop that led to this paper was the first of its kind for
copepods. It was convened to promote international collaboration and to facilitate largescale copepod genomics projects with compelling impact on basic and applied research
problems.
Three overarching themes encompass the diversity of problems that copepod genomics
studies could help to address:
1. Global and Ecosystem Health Copepods are sensitive indicators of local
and global climate change, provide key ecosystem services including the transfer of
carbon between trophic levels and to carbon sinks, and are vectors of human disease (e.g.
cholera).
2. Economy Copepods are a vital food source, which sustain the world’s key
wild and cultured fin-fish stocks. These in turn are critically important to local and
regional economies and subsistence, particularly in developing nations. Copepods are
also major parasites of economically important fish species.
3. Fundamental Biological Understanding Copepods possess features, which
make them uniquely suitable for research that addresses both applied problems as well as
questions about key phenomena such as adaptive radiation, oligomerization, genome
reorganization, mechanisms of diapause, developmental biology, symbiotic relationships
including host-pathogen interactions, and habitat shifts during species invasions.
The exceptional diversity seen within the subclass Copepoda is exemplified by
the widely divergent morphologies, physiologies, life-strategies, and habitat preferences
of its members. Such diversity argues for targeting several copepod species in parallel for
the development of genomics resources. Any individual species, or indeed order, would
certainly fail to be representative of the diversity of the taxon and to encompass the many
biological attributes identified in our three themes. Affordable comparative genomics,
utilizing several copepod taxa and a combination of whole genome and EST library
analysis is now feasible due to recent advances in sequencing technology that have
2
dramatically reduced costs and shortened sequencing times. Our goal is to develop
genomic resources for copepods that facilitate research on problems of global
importance.
The copepod research community is highly eclectic with respect to the species
that are studied and the questions that are asked. This document takes into account this
extraordinary diversity and is designed to illustrate the considerable breadth of problems
which could be addressed using genomics resources of copepods.
... If stress persists, exhaustion occurs whereby the plant shows chronic damage or death. (Modified from Steinberg et al. 2008) that chronic distress dominates any acquired resistance. In this generalized model of stress, improved stress resistance in response to the initial stress involves energetic costs and changes and the expression of different genes to trigger a suite of acclimation processes (e.g., heat shock proteins, osmoregulatory compounds) that enhance resistance to subsequent stress (Steinberg et al. 2008). ...
... (Modified from Steinberg et al. 2008) that chronic distress dominates any acquired resistance. In this generalized model of stress, improved stress resistance in response to the initial stress involves energetic costs and changes and the expression of different genes to trigger a suite of acclimation processes (e.g., heat shock proteins, osmoregulatory compounds) that enhance resistance to subsequent stress (Steinberg et al. 2008). If the stress is maintained, exhaustion eventuates, causing chronic damage and a collapse of cellular functions (e.g., membrane integrity, photosynthetic apparatus). ...
Tree species are exposed to single and combined forms of stress capable of inducing severe changes in plant functioning and survival. Climate change and other human disturbance continue to introduce novel combinations of stressors in forest ecosystems that make predicting their impact exceedingly difficult. In this chapter, we examine the causes and consequences of combined stresses in forest ecosystems. We discuss the significance of a range of abiotic and biotic factors responsible for various impacts on forest ecosystems, including long-term decline and episodic forest collapse. A generalized framework is presented that helps elucidate the contributions of primary, secondary, conditioning, and anthropogenic factors in determining levels of physiological stress in trees. The intensity, frequency, and duration of the constitutive stressors can determine the effect of other stress factors, thereby mediating the importance of singular and multiple factors in defining physiological distress and recovery. The importance of understanding the mechanistic basis for observed stress responses is discussed in light of the challenges associated with predicting impacts from multiple stressors.
... Letztlich ist auch die adaptive Stressantwort als singulärer Erklärungsansatz fragwürdig. Steinberg et al. (2008) führen verschiedene Arbeiten auf, in denen distinkt veränderte Genexpressionsprofile bei Verwendung von hormetisch wirksamen Dosen im Vergleich zu höheren Konzentrationen gefunden wurden. Bei einer rein adaptiven Stressantwort sollten hohe und niedrige Wirkstoffkonzentrationen zwar zu unterschiedlichen Expressions-Intensitäten führen, jedoch nicht zu einer qualitativen Veränderung. ...
... Interessant ist auch, dass diverse Gene als auch die Gengruppe abu durch die lebensverlängernden Konzentrationen in anderer Weise reguliert wurden, als durch die lebens-verkürzende Konzentration. Dies stützt die Hypothese von Steinberg et al. (2008), in der die adaptive Stressantwort als hormetischer Hauptmechanismus angezweifelt wird. Catechin und Gallussäure bewirkten weder bei der Lebensdauer noch bei der Stressresistenz eine Dosis-Wirkungs-Beziehung, die dem Prinzip der Hormesis entspricht. ...
Tannine sind pflanzliche, polyphenolische Sekundärmetabolite mit Protein-präzipitierenden und –bindenden Kapazitäten, welche offenbar für die antinutritiven und gesundheitsschädlichen Wirkungen der Tannine verantwortlich sind. Jedoch dominieren in der Literatur die gesundheitsfördernden Beschreibungen, für die meist die antioxidative Kapazität verantwortlich gemacht wurde. Der etablierte Modelorganismus Caenorhabditis elegans wurde zur Analyse dieses Kontrastes ausgewählt. Zunächst wurden Lebensdauer und Stressresistenz der mit Tanninsäure und den Tanninbausteinen Gallussäure, Ellagsäure und Catechin behandelten Nematoden ermittelt. Das vermutete Vorliegen eines „Calorie Restriction“ (CR)-Effektes als auch einer hormetischen Dosis-Wirkungs-Beziehung wurde überprüft. Weiterhin wurden antimikrobielle und antioxidative Eigenschaften als potentielle Ursachen der Langlebigkeit untersucht und die Auswirkungen auf die Fitness und Genexpression analysiert. Die Einbeziehung verschiedener Alterungs-Theorien rundete die Analyse der Tanninwirkung ab. Alle Tannin-Substanzen konnten eine Lebensverlängerung erwirken, doch erstaunlicherweise erwiesen sich Langlebigkeit und Stressresistenz als individuelle, nicht zwangsläufig gekoppelte Parameter. Hormesis, CR-nachahmende Eigenschaften, antimikrobielle Kapazitäten als auch verschiedene stressrelevante Gene sind teilweise für die Lebensverlängerung verantwortlich. Die antioxidative Kapazität scheint jedoch irrelevant zu sein. Die differentielle Expression mehrerer hundert Gene durch Tanninsäure unterstreicht zudem die Komplexität der Wirkweise. Hinweise zur Bestätigung der „Disposable Soma Theory“, der „Mitohormesis“-Theorie und der „Green Theory of Ageing“ konnten zum Teil gefunden werden. Diese Arbeit zeigt die individuelle und kontrastreiche Wirksamkeit der Tannine auf. Sie unterstreicht, dass Tannine molekulare Regulatoren sind, welche nicht nur auf ihre antioxidative und antinutritive Kapazität reduziert werden sollten.
... If stress persists, exhaustion occurs whereby the plant shows chronic damage or death. (Modified from Steinberg et al. 2008) that chronic distress dominates any acquired resistance. In this generalized model of stress, improved stress resistance in response to the initial stress involves energetic costs and changes and the expression of different genes to trigger a suite of acclimation processes (e.g., heat shock proteins, osmoregulatory compounds) that enhance resistance to subsequent stress (Steinberg et al. 2008). ...
... (Modified from Steinberg et al. 2008) that chronic distress dominates any acquired resistance. In this generalized model of stress, improved stress resistance in response to the initial stress involves energetic costs and changes and the expression of different genes to trigger a suite of acclimation processes (e.g., heat shock proteins, osmoregulatory compounds) that enhance resistance to subsequent stress (Steinberg et al. 2008). If the stress is maintained, exhaustion eventuates, causing chronic damage and a collapse of cellular functions (e.g., membrane integrity, photosynthetic apparatus). ...
Environmental stress can be viewed as the physical, chemical, and biological con-straints on the productivity and development of ecosystems. For plants, Grime (1977) hypothesized that stress is one of the three fundamental drivers shaping plant strategies and he defined stress as a set of external constraints limiting the rate of resource acquisition, growth, or reproduction (Grime 1977). Stress, in a broad sense, is the major force limiting species distribution and ecosystem structure and function. Forest ecosystems are maintained in a dynamic equilibrium by continu-ous stress-inducing factors, as well as stochastic disturbance events. For example, primary climatic stress factors can be broadly categorized as light, temperature, and water and largely explain the distribution of biomes and forest types globally (Boisvenue and Running 2006). Competitive and other biotic interactions are also important in limiting species and population distribution and function. Thus, the role of stress in triggering and shaping plant functioning is complex and can be bet-ter understood by considering responses that arise when a particular individual or population is exposed to conditions outside its normal operating range. The impacts of stress on growth and development are evident at different tem-poral scales for both individuals and populations (Fig. 11.1a). At the whole-plant level, the initial stress response or period of decline in a process such as growth or photosynthesis, happens within seconds to days. Acclimation can follow the ini-tial response and involves compensation or enhanced resistance to the initial stress
... Changes in gene expression are known to reflect organism responses to environmental challenges. They likely allow the detection of potentially hazardous effects, even before measurable reductions of vital activity and cell deterioration, also providing mechanistic information (Robbens et al., 2007;Steinberg et al., 2008). Among the (eco)toxicological studies based on gene expression profiling in diverse organisms (for a revision, see Steinberg et al., 2008), a number have used the microbial eukaryotic model Saccharomyces cerevisiae and unraveled toxicity mechanisms of xenobiotics (Kim et al., 2006;Gil et al., 2011Gil et al., , 2014Yasokawa and Iwahashi, 2010). ...
... They likely allow the detection of potentially hazardous effects, even before measurable reductions of vital activity and cell deterioration, also providing mechanistic information (Robbens et al., 2007;Steinberg et al., 2008). Among the (eco)toxicological studies based on gene expression profiling in diverse organisms (for a revision, see Steinberg et al., 2008), a number have used the microbial eukaryotic model Saccharomyces cerevisiae and unraveled toxicity mechanisms of xenobiotics (Kim et al., 2006;Gil et al., 2011Gil et al., , 2014Yasokawa and Iwahashi, 2010). The yeast has been widely used as a test organism, since it is nonpathogenic, simple and easy to cultivate, has a fully annotated genome, and shares a strong conservation, at both metabolic and regulatory levels, with experimentally less accessible eukaryotes (Daniel et al., 2004;Papaefthimiou et al., 2004;Knight, 2008). ...
The present study is aimed at evaluating whether a gene expression assay with the microbial eukaryotic model Saccharomyces cerevisiae could be used as a suitable warning tool for the rapid preliminary screening of potential toxic effects on organisms due to scenarios of soil and water contamination with pyrimethanil. The assay consisted of measuring changes in the expression of the selected pyrimethanil-responsive genes ARG3 and ARG5,6 in a standardized yeast population. Evaluation was held by assessing the toxicity of surface runoff, a major route of pesticide exposure in aquatic systems due to non-point-source pollution, which was simulated with a pyrimethanil formulation at a semifield scale mimicking worst-case scenarios of soil contamination (e.g. accident or improper disposal). Yeast cells 2-h exposure to the runoff samples led to a significant 2-fold increase in the expression of both indicator genes. These results were compared with those from assays with organisms relevant for the aquatic and soil compartments, namely the nematode Caenorhabditis elegans (reproduction), the freshwater cladoceran Daphnia magna (survival and reproduction), the benthic midge Chironomus riparius (growth), and the soil invertebrates Folsomia candida and Enchytraeus crypticus (survival and reproduction). Under the experimental conditions used to simulate accidental discharges into soil, runoff waters were highly toxic to the standard test organisms, except for C elegans. Overall, results point out the usefulness of the yeast assay to provide a rapid preview of the toxicity level in preliminary screenings of environmental samples in situations of inadvertent high pesticide contamination. Advantages and limitations of this novel method are discussed.
... Regarding fish, they are the most attractive group investigated for gene expression at a highly advanced level, and with a high number of chemical/pharmaceutical stress elements such as metals, metalloids, nanoparticles, single organic compounds, endocrine disrupting compounds, mixture toxicity, effluent toxicity, and sediment toxicity, which affect metabolism, fertilization, cellular processes, amino acid synthesis, apoptosis, among others [111]. Certainly, genomics deepens insights into eco-evolutionary dynamics at the population or community levels because the gene expressions that are significantly up-or down-regulated show a link to the evolutionary process of the defense of a given trait [112]. ...
Freshwater ecosystems face various threats, especially in recent decades, that pose unprecedented challenges to human health, water supply, agriculture, forestry, ecology, and biodiversity. Although progress has been made in biomonitoring techniques tailored to specific countries and communities, significant constraints exist in assessing and quantifying biodiversity and its interaction with detrimental factors. The incorporation of modern techniques into biomonitoring is challenging, with multiple perspectives. This reviewaims to provide a comprehensive overviewof contemporary advancements in freshwater biomonitoring, focusing on omics methodologies such as genomics, metagenomics, transcriptomics, proteomics, metabolomics, and multi-omics. Additionally, this work highlights the need
for modernization by presenting case studies, examining studied organisms, and evaluating the benefits and drawbacks of these methodologies. The utilization of advanced high-throughput bioinformatics techniques represents a departure from conventional practices, necessitating a significant shift. The contributions of omics techniques to biological quality elements (BQEs) and their interpretations of ecological problems are crucial for biomonitoring programs. These contributions identify interactions between different levels of biological organization and their responses to specific critical conditions.
... 110 Furthermore, Western blotting, dot blotting, and immunohistochemistry assays have also been described for the detection of WSSV infections using monoclonal antibodies. 111−113 Microarray technology has instead been used to study environmental science 114 and genetic divergence 115 in aquaculture. Microarrays have also provided an understanding of the regulation of defense mechanisms in aquaculture animals in response to various stimuli. ...
The shrimp aquaculture industry has seen substantial growth over the past two decades, which is driven by the increasing global demand for food. Farmed shrimp products are a highly valued commodity, with Indian market revenue from this industry alone being more than 10 billion USD.
With this growth, the importance of addressing diseases caused by infectious agents such as bacteria, viruses, fungi, and protozoa has become increasingly important. Additionally, the mismanagement of antibiotics and the rise of antimicrobial resistance pose significant challenges in the industry.
Efficient diagnostic solutions are needed to address these challenges and protect the sustainability of industry and consumer health. While traditional methods are used for detecting culturable pathogens, molecular diagnostics are still in their early stages of development. The high cost of molecular testing and the
limited availability of laboratories are some of the challenges faced by aquaculture farmers. The development of rapid, sensitive, and deployable diagnostics and research tools at the farm and hatchery levels is essential for a better understanding and management of diseases and antimicrobial resistance.
The importance of diagnostics in shrimp aquaculture cannot be overstated, as it is essential for the sustainability of the industry and protection of consumer health. In addition to continued research, industry must also focus on the commercialization of these technologies, as commercialized diagnostic solutions can offer a standardized and trustworthy method of disease management. This, in turn, can result in better disease control and a more sustainable shrimp aquaculture industry.
... At present, high-throughput "omics" technologies are enabling ecotoxicological studies to successfully derive toxicity data at molecular, cellular and organism levels (Ebner, 2021;Steinberg et al., 2008). However, the importance of genome-wide changes in genetic diversity, variations in allelic or genotypic frequencies and genome structure of affected populations has received less attention (Bickham, 2011;Dallinger and Höckner, 2013). ...
Susceptibility to hazardous materials and contamination is largely determined by genetic make-up and evolutionary history of affected organisms. Yet evolutionary adaptation and microevolutionary processes triggered by contaminants are rarely considered in ecotoxicology. Using an evolve and resequencing approach, we investigated genome-wide responses of the midge C. riparius exposed to virgin polyamide microplastics (0–180 μm size range, at concentration 1 g kg⁻¹) during seven consecutive generations. The results were integrated to a parallel life-cycle experiment ran under the same exposure conditions. Emergence, life-cycle trait, showed first a substantial reduction in larval survival, followed by a rapid recovery within three generations. On the genomic level, we observed substantial selectively driven allele frequency changes (mean 0.566 ± 0.0879) within seven generations, associated with a mean selection coefficient of 0.322, indicating very strong selection pressure. Putative selection targets were mainly connected to oxidative stress in the microplastics exposed C. riparius population. This is the first multigenerational study on chironomids to provide evidence that upon exposure to polyamide microplastic there are changes on the genomic level, providing basis to rapid adaptation of aquatic organisms to microplastics.
... Gene expression can be influenced when direct or indirect exposure to various environmental pollutants and stressors in organisms (Steinberg et al., 2008). Cytochrome P450 (CYP450) enzyme system are involved in the metabolism process of xenobiotics and CYP360A8 in invertebrates is a homologous gene to CYP3A in mammals that plays a vital role in various oxidative metabolism and detoxification of contaminants in vivo (Liu et al., 2017;Liu et al., 2019;Bao et al., 2020). ...
The drastic increase of microplastics (MPs) in aquatic environment has become a serious threat to marine and freshwater ecosystems. However, little information is available regarding the potential detrimental effects of polyvinyl chloride microplastics (PVC-MPs) on aquatic organisms. This study investigated the changes of reproduction parameters, oxidative stress and the expression of reproduction and detoxification-related genes in Daphnia magna after exposed to 2 ± 1 and 50 ± 10 μm PVC-MPs. The results showed that chronic exposure to 2 ± 1 μm PVC-MPs prolonged days to the first brood, increased total number of broods per female and frequency of molting per adult, decreased offspring number at first brood and total number of offspring per female in D. magna. Moreover, 2 ± 1 μm PVC-MPs also disturbed the activities of SOD and CAT, increased GSH and MDA levels. The expression of Vtg, SOD, CAT, CYP314 and CYP360A8 genes also exhibited different response patterns depending on exposure time. Furthermore, 50 ± 10 μm PVC-MPs decreased offspring at first brood and Vtg mRNA level, increased the transcription levels and activities of SOD and CAT. These results suggest that the presence of PVC-MPs in aquatic environment may cause reproduction toxicity by disrupting the reproduction and detoxification-related genes expression and inducing oxidative stress in D. magna.
... The detection of transcriptional profiles has been proposed as an approach that could provide insights into, and facilitate risk assessment of, actual and potential damage caused by environmental stress. Such tools could be employed as early and sensitive warning systems of environmental contaminants or change before the impacts are discernible at the population level in biological communities (Clements 2000;Steinberg et al. 2008;Fedorenkova et al. 2010;Lauritano et al. 2012;Marinkovic et al. 2012;Mantilla et al. 2018). Here, we focus on transcriptional responses of genes that mediate defense mechanisms in aquatic invertebrates and classify the types of stressors involved into physical factors and chemical compounds, based on the results of studies that have been conducted on aquatic chironomids from 1991 to 2020. ...
In freshwater ecosystems, aquatic invertebrates are influenced continuously by both physical stress and xenobiotics. Chironomids (Diptera; Chironomidae), or non-biting midges, are the most diverse and abundant invertebrates in freshwater habitats. They are a fundamental link in food chains of aquatic ecosystems. Chironomid larvae tolerate stress factors in their environments via various physiological processes. At the molecular level, environmental pollutants induce multi-level gene responses in Chironomus that regulate cellular protection through the activation of defense processes. This paper reviews literature on the transcriptional responses of biomarker genes to environmental stress in chironomids at the molecular level, in studies conducted from 1991 to 2020 (120 selected literatures of 374 results with the keywords “Chironomus and gene expression” by PubMed search tool). According to these studies, transcriptional responses in chironomids vary depending on the type of stress factor and defensive responses associated with antioxidant activity, the endocrine system, detoxification, homeostasis and stress response, energy metabolism, ribosomal machinery, apoptosis, DNA repair, and epigenetics. These data could provide a comprehensive overview of how Chironomus species respond to pollutants in aquatic environments. Furthermore, the transcriptomic data could facilitate the development of genetic tools for water quality and environmental monitoring based on resident chironomid species.
... Further, C. elegans has been used to study transgenerational and environmental epigenetics (Kelly 2014;Weinhouse et al. 2018). C. elegans is now a wellestablished model for human and ecological toxicology employed by many labs (a non-comprehensive sampling identifies approximately two dozen: (Leung et al. 2008;Boyd et al. 2010;Steinberg, Sturzenbaum, and Menzel 2008;Helmcke, Avila, and Aschner 2010;Meyer and Williams 2014;Tejeda-Benitez and Olivero-Verbel 2016;Hunt 2017;Choi et al. 2014;Honnen 2017;Ferreira and Allard 2015;Allard et al. 2013;Lenz, Pattison, and Ma 2017;Nass, Miller, and Blakely 2001a;Harrington et al. 2010;Cooper and Van Raamsdonk 2018;Liao and Yu 2005;Fitsanakis, Negga, and Hatfield 2014;Menzel et al. 2005;Harlow et al. 2018;Zhao et al. 2013;Brady et al. 2019;Anbalagan et al. 2013;Clavijo et al. 2016;Hoss et al. 2009;Horsman and Miller 2016;Shomer et al. 2019;Zhang et al. 2020;Haegerbaeumer et al. 2018b;Lee et al. 2020;Shen et al. 2019;Harlow et al. 2016;Dietrich et al. 2016;Xiong, Pears, and Woollard 2017)). The number of publications on toxicology and related fields in C. elegans has grown rapidly in recent years, with an even more rapid growth in pharmacology-related publications ( Figure. ...
Caenorhabditis elegans has emerged as a major model in biomedical and environmental toxicology. Numerous papers on toxicology and pharmacology in C. elegans have been published, and this species has now been adopted by investigators in academic toxicology, pharmacology, and drug discovery labs. C. elegans has also attracted the interest of governmental regulatory agencies charged with evaluating the safety of chemicals. However, a major, fundamental aspect of toxicological science remains underdeveloped in C. elegans: xenobiotic metabolism and transport processes that are critical to understanding toxicokinetics and toxicodynamics, and extrapolation to other species. The aim of this review was to initially briefly describe the history and trajectory of the use of C. elegans in toxicological and pharmacological studies. Subsequently, physical barriers to chemical uptake and the role of the worm microbiome in xenobiotic transformation were described. Then a review of what is and is not known regarding the classic Phase I, Phase II, and Phase III processes was performed. In addition, the following were discussed (1) regulation of xenobiotic metabolism; (2) review of published toxicokinetics for specific chemicals; and (3) genetic diversity of these processes in C. elegans. Finally, worm xenobiotic transport and metabolism was placed in an evolutionary context; key areas for future research highlighted; and implications for extrapolating C. elegans toxicity results to other species discussed.
... The nematode Caenorhabditis elegans is a model organism widely used to study toxicogenic responses to environmental pollutants at different multi-levels of biological complexity, from molecular to organism (Steinberg et al. 2008;Polak et al. 2014). One of the strengths of this model is the availability of mutants that allow to effectively investigate the molecular mechanisms that underlie cellular and organism responses (Qabazard et al., 2013;Clavijo et al. 2016;Imanikia et al. 2016;Gajski et al. 2019;Queirós et al. 2019). ...
Bisphenol F (BPF) and bisphenol S (BPS) have been developed as an alternative to bisphenol A (BPA), a well-known endocrine disruptor, leading to their detection in the aquatic environment. In this work, we used the animal model Caenorhabditis elegans to improve our understanding of their potential effects on the biota and the environment. Our findings demonstrated that, after 24 h exposure, all the bisphenols examined increased the number of apoptotic corpses and the expression of the detoxifying enzymes SOD-3 and GST-4, without affecting the ROS levels, while BPA and BPS significantly enhanced DNA fragmentation. Furthermore, similarly to BPA, BPF and BPS did not alter the lifespan through the activation of SEK-1 and SKN-1 pathways. Thus, this study raises the attention of the risks associated with exposure to BPA alternatives. We also examined the effects of microplastic (MP) eluates on C. elegans. Aqueous extracts of weathered microplastic samples, both at high and low degradation state and pellets, have been evaluated for their effects on lifespan, DNA fragmentation, germline apoptosis, and oxidative stress response. Overall, our findings showed that eluates of low degraded plastics exert a greater toxic effect on the nematode C. elegans compared with the aqueous sample of high degraded plastic fragments and pellets.
... The movement of pesticides into water bodies can occur through subsurface drainage, leaching, run-off, and spray drift (Cosgrove et al., 2019). These compounds can cause biochemical and physiological changes in both non-target organisms and humans, inducing different toxicity mechanisms (Correia et al., 2003;Lushchak et al., 2018;Steinberg et al., 2008;Zhou et al., 2019). ...
Freshwater ecosystems are constantly threatened by the advance of agricultural activities. Abiotic variables (such as temperature, ammonia, and nitrite) and contaminants (e.g. pesticides) can potentially interact, increasing metabolism and the absorption of toxic substances, which can alter the ability of organisms to establish adequate stress responses. This study aimed to verify which pesticides were most frequently found and in the greatest quantities in low-order streams, and whether the combination of these pesticides with the abiotic variables altered the biological metabolism of aeglids. These freshwater crustaceans are important shredders that inhabit low-order streams and are sensitive to disturbances and/or abrupt environmental variations. The animals were exposed in situ in four streams (reference site and sites 1, 2, and 3). The reference site is a preserved stream with no apparent anthropogenic interference where aeglids still occur, while the other sites no longer exhibit populations of these animals and are influenced by agricultural activities. The exposure was performed bimonthly from November 2017 to September 2018 and lasted 96 h. Measured abiotic data and water samples were collected through all days of exposure. The analyzed biochemical parameters were acetylcholinesterase activity in muscle; and glutathione S-transferase, lipid peroxidation, protein carbonylation, non-protein thiols, antioxidant capacity against peroxides, and reactive oxygen species (ROS) in muscle, gills, and hepatopancreas. We found 24 active principles of pesticides, the most frequently being clomazone, atrazine, and propoxur. Bentazone was present at the highest amounts. The parameters evaluated in this study, including biochemical biomarkers and abiotic factors measured from the water, provided a separation of the months as a function of environmental conditions. There was a difference in activity and biomarker levels throughout the year within the same site and in some months between sites. The greater concentration or variety of pesticides associated with extreme abiotic (very high temperatures) data generated increased oxidative stress, with high levels of protein damage and considerable lipid damage in all tissues, as well as elevation in ROS, even with high levels of antioxidant capacity and non-protein thiols. With these data, we intend to warn about the risks of exposure to these environmental conditions by trying to contribute to the preservation of limnic fauna, especially aeglid crabs, because most species are under some degree of threat.
... Les tests écotoxicologiques réalisés ne sont cependant peut-être pas assez sensibles pour détecter ces effets. En effet, les sous-produits issus de l'ozonation sont bien souvent polaires et ne sont donc pas bien extraits lors de la concentration des échantillons (pour les tests in vitro) (Stalter, et al., 2010c). L'ozonation et le traitement CAP-UF restent néanmoins des traitements efficaces contre les micropolluants. ...
Nowadays, micropollutants problem is one of the big issues of decontamination of freshwater in the world. Numerous treatments called "tertiary treatments" begin to be installed in waste water treatment plant. Apart from this aspect, we need to develop toxicity measurement procedures to know the impact of micropollutants on the environment. In addition, this will lead to characterize contaminant removal after treatment. A large panel of bioassay used in waste water treatment plant was established. We can extract 13 tests the most used because of their ease of implementation, their low price, their sensitivity, their repeatability and their reliability : Daphnia death (ISO 6341) ; growth inhibition of phytoplankton (ISO 10253) ; growth inhibition of aquatic plant ; bioluminescence inhibition (ISO 11348-3) ; fish larvae ; fish ; sea urchins ; MTT test (cytotoxicity) ; Ames test (genotoxicity) ; Yas test (ED) ; YES test (ED) ; ER test (ED) ; AR test (ED). Along with this study, two bioassays were carried out on samples from tertiary treatment of coupling ozonation/nanofiltration: Daphnia magna Straus test and Thamnocephalus platyurus test. 6 micropollutants were analysed: carbamazepin (CBZ), acetaminophen (ACT), sulfamethoxazole (SML) ; diclofenac (DCF), Tetracycline (TET), Terbutryn (TER). These organic compounds were treated individually for O3 and mixed for NF. Total concentration of micropollutants was lower than 6mg/l inputs each process. Nanofiltration removal was higher than 80% for each compound. On the whole, no significative effect was observed on Daphnia magna straus on this process. For ozonation, samples of TER and DCF brought out a large death of daphnia after 48h exposure (>30%) input and output of the treatment. Thamnocephalus platyurus test showed the same trend on ozonation process with higher values: >90% of death after 48h exposure. This last test seems the most suitable at these concentrations. Technically, Thamnocephalus platyrus test was the easiest to set up (24h for hatching eggs) despite size difference of organisms.
... Organisms respond to environmental contaminants and stressors by altering their gene expression profiles as a direct or indirect result of exposure (Steinberg et al., 2008). Understanding gene expression analysis associated to physiological and phenotypic alterations can provide further insight into the molecular mechanisms of toxicity. ...
Phthalates are used as plasticizers to increase durability, resistivity and flexibility of plastic materials. The commonly used phthalate, diethylhexyl phthalate (DEHP) is used in different plastic materials like food packaging, toys and medical devices. DEHP has been linked to different toxicities in humans as well as in animals, and as a consequence other phthalates, including dibutyl phthalate (DBP) and diethyl phthalate (DEP) are being introduced. The increased use of phthalates has resulted in contamination of aquatic ecosystem and it directly threatens the aquatic life. In this study, we analyzed the effects of three phthalates DEHP, DEP and DBP using freshwater organism Daphnia magna. Although, exposure of the three phthalates at 1 and 10 μM did not result any lethality and hatching delay, the chronic exposure for 14 days resulted in reduction of body length. There was enhanced fat accumulation on exposure to all the phthalates, as indicated by oil red O staining. qRT-PCR analysis of genes involved in fat metabolism suggests that the increase in fat content could be due to inhibition of absorption and catabolism of fatty acids. Reproduction analysis showed that DBP and DEP did not alter fecundity but surprisingly, DEHP at 1 μM increased reproduction by 1.5 fold compared to control group. Phthalates also showed negative effect on lifespan as DEP at 10 μM and DBP at both 1 and 10 μM significantly reduced the lifespan. Our data indicates that along with the banned phthalate DEHP, the other substitute phthalates DEP and DBP could also have detrimental effect on aquatic organisms.
... Diverse aquatic organisms have been examined via response monitoring, and the usefulness of several biological parameters as biomarkers has consequently been proven (Bradbury et al., 1991;Ankley et al., 2007;Segner, 2009;Volz et al., 2011;Jeong et al., 2014). Recent advances in omics techniques allow the acquisition of high-throughput data and have widened the platforms for sub-organismal response monitoring (Dzialowski et al., 2006;Poynton et al., 2008;Steinberg et al., 2008;Nestler et al., 2012). In case of active pharmaceutical ingredients (APIs), whose pharmacological and toxicological information is well established in the drug discovery and development stages, the monitoring data is usually compared with their established background data, using which their MOA in model organisms is deduced (Escher and Hermens, 2002;Escher et al., 2005;Campos et al., 2012). ...
Studies are underway to gather information about the mode of action (MOA) of emerging pollutants that could guide practical environmental decision making. Previously, we showed that propranolol, an active pharmaceutical ingredient, had adverse effects on Daphnia magna that were similar to its pharmaceutical action. In order to characterize the mode of action of propranolol in D. magna, which is suspected to be organ-specific pharmaceutical action or baseline toxicity, we performed time-series monitoring of behavior along with heart rate measurements and nuclear magnetic resonance (NMR) based metabolite profiling. Principle component analysis (PCA) and hierarchical clustering were used to categorize the mode of action of propranolol among 5 chemicals with different modes of action. The findings showed that the mode of action of propranolol in D. magna is organ-specific and vastly different from those of narcotics, even though metabolite regulation is similar between narcotic and non-narcotic candidates. The method applied in this study seems applicable to rapid characterization of the MOA of other cardiovascular pharmaceutical ingredients.
... Hence, the exploration of haemocyte-expressed genes profile can provide a dynamic view of biological processes, to allow the correct sorting of different functional states [46], and understand the role of genetic basis in mussel resistance or susceptibility to infection to identify the causes of mortalities and the traits that characterize enhanced survival capacities in mussel populations [43], creating a conceptual bridge between the early organism responses and late population changes [47]. ...
... Hence, the exploration of haemocyte-expressed genes profile can provide a dynamic view of biological processes, to allow the correct sorting of different functional states [46], and understand the role of genetic basis in mussel resistance or susceptibility to infection to identify the causes of mortalities and the traits that characterize enhanced survival capacities in mussel populations [43], creating a conceptual bridge between the early organism responses and late population changes [47]. ...
The Mediterranean mussel Mytilus galloprovincialis is an ecologically and economically important species. It has been used in programs of monitoring of pollutions, since it is sessile organism that is capable of accumulating pollutants in tissues through filter feeding. Due to an increase of pollutants in the environment, marine mussels present physiological alterations that compromise their innate immune system, which can latter lead to opportunistic diseases. The haemocytes are the cells in charge of the immune response in the Mediterranean mussel and in other mollusks. In this review, we summarize the physiological and genetic response capacity of these immune cells to the presence of xenobiotics, pathogens and the interplay. The identification of the basic mechanisms of immunity and their modulation in mussels can give important information for the possible utilization of this species as an invertebrate model for studies on innate immunity, future immunotoxicological studies, and predict changes in the community for the future.
... The field is relatively new, accounting for the fact that little research with this particular species has been carried out with emphasis on the relationship between environmental exposures and genetics. Nevertheless, considering that C. elegans mimics toxicological outcomes in mammals (Helmcke et al., 2009(Helmcke et al., , 2010Leung et al., 2008;Steinberg et al., 2008) and given the need for alternative models, this model will likely become more prevalent in years to come. Furthermore, as C. elegans readily and easily allows for the use of high throughput techniques, screening for several toxicants will become faster and more consistent. ...
It is widely accepted that the developing nervous system is especially vulnerable to a variety of chemicals, including drugs and environmental contaminants. It is also clear that our understanding of the risks from chemical exposures during development is rudimentary, and that the resources for remedying the situation are legion. As a result, increasing attention is directed toward alternative test methods including in vitro preparations, computational (in silico) models, and in vivo model (or alternative) organisms. In particular, zebrafish have become a popular test species in toxicology, pharmacology, and biomedical research. In this chapter, we review several issues, results, and research needs regarding the use of zebrafish to assess developmental neurotoxicity. Considerable advances have been made in understanding the basic biology of nervous system development in zebrafish, in techniques for rapidly evaluating the effect of chemical exposures on nervous system development, and, notably, to a lesser extent, in understanding the significance of results for predicting human effects. This chapter was written as an introduction to the use of zebrafish in developmental neurotoxicology, and to encourage the use of this model either for screening or mechanistic purposes. We have endeavored to make the reader aware of significant research findings, and to offer a balanced view of the advantages and limitations in using zebrafish as a model for investigating developmental neurotoxicity.
... mediated by stress hormones-corticosteroids) provoking mobilization of the endogenous resources to restore homeostasis (contra-shock). During the resistance stage, the cell (organism) restores homeostasis and acquires resistance that lasts until resources are exhausted [1,2]. ...
The ability to adapt to adverse conditions permits many bacterial species to be virtually ubiquitous and survive in a variety of ecological niches. This ability is of particular importance for many plant pathogenic bacteria that should be able to exist, except for their host plants, in different environments e.g. soil, water, insect-vectors etc. Under some of these conditions, bacteria encounter absence of nutrients and persist, acquiring new properties related to resistance to a variety of stress factors (cross-protection). Although many studies describe the phenomenon of cross-protection and several regulatory components that induce the formation of resistant cells were elucidated, the global comparison of the physiology of cross-protected phenotype and growing cells has not been performed. In our study, we took advantage of RNA-Seq technology to gain better insights into the physiology of cross-protected cells on the example of a harmful phytopathogen, Pectobacterium atrosepticum (Pba) that causes crop losses all over the world. The success of this bacterium in plant colonization is related to both its virulence potential and ability to persist effectively under various stress conditions (including nutrient deprivation) retaining the ability to infect plants afterwards. In our previous studies, we showed Pba to be advanced in applying different adaptive strategies that led to manifestation of cell resistance to multiple stress factors. In the present study, we determined the period necessary for the formation of cross-protected Pba phenotype under starvation conditions, and compare the transcriptome profiles of non-adapted growing cells and of adapted cells after the cross-protective effect has reached the maximal level. The obtained data were verified using qRT-PCR. Genes that were expressed differentially (DEGs) in two cell types were classified into functional groups and categories using different approaches. As a result, we portrayed physiological features that distinguish cross-protected phenotype from the growing cells.
... The PWS1 and KOD groups appeared to have immunological or physiological responses that indicated greater organic compound exposure relative to the other populations examined, but their profile motifs differed, suggesting unique environmental stressors at each site. Genomic profiling has successfully linked specific signatures to unique combinations of chemical contaminants in other species [50][51][52][53]. In fact, the transcription profile of the KOD otters is more consistent with that of a dioxin-induced profile, while the transcription profile of PWS1 otters (in particular, those from the area that received the heaviest shoreline oiling in 1989) is more consistent with a polycyclic aromatic hydrocarbon (PAH)-induced profile [27]. ...
Using a panel of genes stimulated by oil exposure in a laboratory study, we evaluated gene transcription in blood leukocytes sampled from sea otters captured from 2006–2012 in western Prince William Sound (WPWS), Alaska, 17–23 years after the 1989 Exxon Valdez oil spill (EVOS). We compared WPWS sea otters to reference populations (not affected by the EVOS) from the Alaska Peninsula (2009), Katmai National Park and Preserve (2009), Clam Lagoon at Adak Island (2012), Kodiak Island (2005) and captive sea otters in aquaria. Statistically, sea otter gene transcript profiles separated into three distinct clusters: Cluster 1, Kodiak and WPWS 2006–2008 (higher relative transcription); Cluster 2, Clam Lagoon and WPWS 2010–2012 (lower relative transcription); and Cluster 3, Alaska Peninsula, Katmai and captive sea otters (intermediate relative transcription). The lower transcription of the aryl hydrocarbon receptor (AHR), an established biomarker for hydrocarbon exposure, in WPWS 2010–2012 compared to earlier samples from WPWS is consistent with declining hydrocarbon exposure, but the pattern of overall low levels of transcription seen in WPWS 2010–2012 could be related to other factors, such as food limitation, pathogens or injury, and may indicate an inability to mount effective responses to stressors. Decreased transcriptional response across the entire gene panel precludes the evaluation of whether or not individual sea otters show signs of exposure to lingering oil. However, related studies on sea otter demographics indicate that by 2012, the sea otter population in WPWS had recovered, which indicates diminishing oil exposure.
... Diagnostic tools for specific groups of chemicals or types of response are needed (Cajaraville et al., 2000). Emerging disciplines such as genomics, proteomics, and biodiversity informatics may provide solutions (Porte et al., 2006;Aricò, 2008;Steinberg et al., 2008;Mearns et al., 2008). ...
... Indeed, biphasic response is a recognized biological phenomenon that is usually observed in the presence of low levels of aquatic contaminants (Hanekamp and Calabrese, 2006;Calabrese, 2013). However, biphasic responses may not be limited to a simple adaptive response, but accompanied by a transcriptional activation of different pathways that may interact with themselves, increasing the level of complexity in toxicological studies (Steinberg et al., 2008). ...
... One central paradigm of hormesis is that the " adaptive response " is characterized by biphasic dose responses that display analogous quantitative features such as the amplitude and range of the stimulatory response (which are either directly induced or the result of compensatory biological processes following an initial disruption in homeostasis) (Calabrese and Baldwin 2002). However several recent papers (reviewed by Steinberg et al. (2008)) have challenged this paradigm and suggest that low and high dose exposure are characterized by different pathways. In order to identify the major regulatory and metabolic pathways, we compared the whole-genome transcriptomes of nematodes exposed to three different polyphenolic substrates with increasing complexity at the molecular and structural level, namely quercetin, tannic acid, and humic substances (HuminFeed ® , HF), a commercial humic preparation. ...
Interactions between plants and herbivores are of major importance in natural ecosystems. The phytochemical co-evolution theory suggests that plant secondary metabolites are likely the most important mediators of plant-herbivore interactions, although their primary cause for production is the shielding effect against adverse environmental triggers. According to this theory, both plants and insect herbivores generate selective forces that lead to the evolution of plant defense (i.e., plant secondary metabolites, PSM) and herbivore offense (i.e., detoxification ability) in a so-called co-evolutionary arms race. These chemicals, although not required for primary plant metabolic processes such as respiration or growth, have been extensively recognized for their role in plant defense against herbivore and pathogen attack (Bidart-Bouzat and Imeh-Nathaniel 2008). There is still some concern, though, as to whether or not the primary reason for the production of PSM is protection from photodamage rather than from herbivory. Nevertheless, there is no doubt that these metabolites also function as food allelochemicals, that is plant defense.
... Changes in gene expression can provide explicit indicators of stress, and have an added advantage of being able to identify some specific physiological causes of stress (Steinberg et al., 2008;Veldhoen et al., 2012;Weirup et al., 2013). Among humans, gene expression profiles are commonly used as biomarkers of diseases or physiological stress (Strand et al., 2005;Abba et al., 2010;Zhou et al., 2012). ...
Gene expression profiles are increasingly being used as biomarkers to detect the physiological responses of a number of species to disease, nutrition, and other stressors. However, little attention has been given to using gene expression to assess the stressors and physiological status of marine mammals. We sought to develop and validate a nutrigenomics approach to quantify nutritional stress in Steller sea lions (Eumetopias jubatus). We subjected 4 female Steller sea lions to 3 feeding regimes over 70-day trials (unrestricted food intake, acute nutritional stress, and chronic nutritional stress), and drew blood samples from each animal at the end of each feeding regime. We then extracted the RNA of white blood cells and measured the response of 8 genes known to react to diet restriction in terrestrial mammals. Overall, we found the genomic response of Steller sea lions experiencing nutritional stress was consistent with diet restriction regulation in terrestrial mammals. Our nutritionally stressed sea lions down-regulated some cellular processes involved in immune response and oxidative stress, and up-regulated pro-inflammatory responses and metabolic processes. Nutrigenomics appears to be a promising means to monitor nutritional status and contribute to mitigation measures needed to assist in the recovery of Steller sea lions and other at-risk species of marine mammals.
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... Steinberg et al., 2008). Furthermore, the assessment of modifications at the molecular and biochemical level mediated by xenobiotics is important for the understanding of the induced stress response (Reichert and Menzel, 2005). ...
Dibromoacetic acid (DBAA) and N-nitrosodimethylamine (NDMA) have natural and anthropogenic
sources and are ubiquitously distributed in the environment. They are classified as toxic and
carcinogenetic and various studies have addressed their effects on vertebrates. Furthermore, there is
no information about the whole-organism effects at low concentrations or about their impact on
invertebrates. Therefore, these compounds were studied with the model invertebrate Caenorhabditis
elegans (C. elegans) at relatively low concentrations. Biological tests (life span, reproduction, body size,
thermal stress resistance) as well as biochemical (pro- and antioxidative capacity and lipid peroxidation)
and biomolecular assays (transcription of stress genes) were performed. None of the applied concentrations
showed a toxic potential. Instead, they extended life span and increased the body length. Both xenobiotics
did not cause oxidative stress or DNA damages, or acted as endocrine disruptors. The stimulatory
effects on C. elegans were most likely not a result of an induced protective stress response. Instead, an
‘energy saving mode’, indicated by the reduced transcription of many stress response genes, could have
provided additional resources for longevity and growth. Although both substances are potentially toxic at
higher doses, the present study underlines the importance of testing lower concentrations and their
impact on invertebrates.
... Ecotoxicogenomics has improved understanding of the linkages between molecular events and whole animal responses (Steinberg et al., 2008). Researchers in the area of ecotoxicology have described a new approach that has high relevance for risk assessment and management that is termed adverse outcome pathways (AOPs). ...
Exposure assessment is a critical step in the risk assessment process and is an important component of dose–response analysis and interpretation of causal associations in epidemiology studies. Exposure assessment describes the nature, magnitude, duration, and extent to which various populations are exposed to a chemical agent. There are several factors to consider when conducting an exposure assessment, such as contaminated media, route of exposure, timing of exposure (duration, frequency), and bioavailability. This chapter will review the general principles of exposure assessment, as well as methods and available tools for evaluating exposures from different media (air, soil, water, food) and different routes of exposure (dermal, oral, inhalation). Additionally, this chapter will review different methods for quantifying exposures, whether through direct measurement, historical reconstruction, modeling, or biomarkers/biomonitoring. Methods for quantifying variability and uncertainty in the exposure assessment through modeling and sensitivity analyses are also discussed. The field of exposure assessment has made significant advancements over the last several decades. The chapter will end on reflections of several lessons learned over the evolution of exposure assessment and insights for its future.
Effective marine management requires comprehensive data on the status of marine biodiversity. However, still there is a void for the efficiency of present methods to document the biodiversity in the aquatic environment. Environmental DNA (eDNA) and proteins sourced from seawater shows a new area for exploring the biota in marine ecosystems. Here, we discuss about the potential of eDNA on the marine biodiversity. Recently, all the fields utilize the genomics and proteomics, particularly in marine aquaculture biodiversity studies, eDNA research and microbiome analysis with the help of computational approaches using specific coding pipelines and configurations. Additionally, macromolecular structures such as DNA or proteins are available in Protein data bank (PDB), the prediction of active sites and excluded volume information used for the field of chemoinformatics. To improve the production of animals and aquaculture industry these type of ligands are used as an immunomodulators in aquatic biotechnology. On this account, this chapter aims to evaluate the bioinformatics applications in aquaculture through various approaches. This chapter will give a wide range of information (about aquatic organisms) of all eDNA and protein from NDB and PDB respectively, this will be helpful for all scientific researchers and scholars working in this avenue. For biodiversity studies we use several sequencing methods to count the number of species and genus type, for instance here we use illlumina sequencing to analyze the eDNA sediments in aquatic environment which provides the biodiversity of various aquatic organisms and monitoring the population density of species in different environments
Accidental spills and the misuse of chemicals may lead to current and legacy environmental contamination and pose concerns over possible (eco)toxicological secondary effects and risks toward non-target microbes and higher eukaryotes, including humans, in ecosystems. In the last decades, scientists and regulators have faced requests to thoroughly screen, prioritize and predict the possible deleterious effects of the huge numbers of existing and emerging xenobiotics, wastewaters and environmental samples on biological systems. In this context, it has become necessary to develop and validate (eco)toxicity bioassays based on microorganisms (e.g., bacteria, microalga, yeast, filamentous fungi, protozoa) as test-organisms whose data should be meaningful for environmental (micro)organisms that may be exposed to contaminated environments. These generally simple, fast and cost-effective bioassays may be preliminary and complementary to the more complex and long-term whole-organism animal-based traditional ecotoxicity tests. With the goal of highlighting the potential offered by microbial-based bioassays as non-animal alternatives in (eco)toxicity testing, the present chapter provides an overview of the current state-of-the art in the development and use of microbial toxicity bioassays through the examination of relatively recent examples with a diverse range of toxicity endpoints. It goes into the (eco)toxicological relevance of these bioassays, ranging from the more traditional microalga- and bacterial-based assays already accepted at regulatory level and commercially available to the more innovative microbial transcriptional profiling and gene expression bioassays, including some examples of biosensors.
The exposome is the cumulative measure of environmental influences and associated biological responses across the life span, with critical relevance for understanding how exposures can impact human health. Metabolomics analysis of biological samples offers unique advantages for examining the exposome. Simultaneous analysis of external exposures, biological responses, and host susceptibility at a systems level can help establish links between external exposures and health outcomes. As metabolomics technologies continue to evolve for the study of the exposome, metabolomics ultimately will help provide valuable insights for exposure risk assessment, and disease prevention and management. Here, we discuss recent advances in metabolomics, and describe data processing protocols that can enable analysis of the exposome. This chapter focuses on using liquid chromatography–mass spectrometry (LC-MS)-based untargeted metabolomics for analysis of the exposome, including (1) preprocessing of untargeted metabolomics data, (2) identification of exposure chemicals and their metabolites, and (3) methods to establish associations between exposures and diseases.
High-throughput mass spectrometry (MS) metabolomics profiling of highly complex samples allows the comprehensive detection of hundreds to thousands of metabolites under a given condition and point in time and produces information-rich data sets on known and unknown metabolites. One of the main challenges is the identification and annotation of metabolites from these complex data sets since the number of authentic standards available for specialized metabolites is far lower than an account for the number of mass spectral features. Previously, we reported two novel tools, MetNet and MetCirc, for putative annotation and structural prediction on unknown metabolites using known metabolites as baits. MetNet employs differences between m/z values of MS1 features, which correspond to metabolic transformations, and statistical associations, while MetCirc uses MS/MS features as input and calculates similarity scores of aligned spectra between features to guide the annotation of metabolites. Here, we showcase the use of MetNet and MetCirc to putatively annotate metabolites and provide detailed instructions as to how those can be used. While our case studies are from plants, the tools find equal utility in studies on bacterial, fungal, or mammalian xenobiotic samples.
The roundworm Caenorhabitis elegans has been an established model organism for the study of genetics and developmental biology, including studies of transcriptional regulation, since the 1970s. This model organism has continued to be used as a classical model system as the field of transcriptional regulation has expanded to include scientific advances in epigenetics and chromatin biology. In the last several decades, C. elegans has emerged as a powerful model for environmental toxicology, particularly for the study of chemical genotoxicity. Here, we outline the utility and applicability of C. elegans as a powerful model organism for mechanistic studies of environmental influences on the epigenome. Our goal in this article is to inform the field of environmental epigenetics of the strengths and limitations of the well‐established C. elegans model organism as an emerging model for medium‐throughput, in vivo exploration of the role of exogenous chemical stimuli in transcriptional regulation, developmental epigenetic reprogramming, and epigenetic memory and inheritance. As the field of environmental epigenetics matures, and research begins to map mechanisms underlying observed associations, new toolkits and model systems, particularly manipulable, scalable in vivo systems that accurately model human transcriptional regulatory circuits, will provide an essential experimental bridge between in vitro biochemical experiments and mammalian model systems. Environ. Mol. Mutagen., 2018. © 2018 Wiley Periodicals, Inc.
A sensitive and selective colorimetric method has been developed for detection of Al³⁺ ion using 4-benzoyl pyrazolone-functionalized gold nanoparticles (BMPBP-AuNPs) as novel colorimetric probes. The BMPBP-AuNPs were characterized by UV-visible spectrometry and transmission electron microscopy (TEM). It was found that the addition of the Al³⁺ ions led to a rapid aggregation of the BMPBP-AuNPs, which changed the color of the mixture from red to blue. Furthermore, there was a shift in the characteristic surface plasmon resonance (SPR) peak from 524 to 650 nm of BMPBP-AuNPs, which confirmed that a good linear relation (R² = 0.9935) was present between the absorption ratio of 524 and 650 nm. Also, the assay detected the Al³⁺ ion concentrations in the linear range 0-12 ppm with the detection limit is 0.05 ppm. Finally, the synthesized BMPBP-AuNPs were successfully used as a colorimetric sensor for the selective and sensitive detection of the Al³⁺ ions in water samples.
Two groups of Apostichopus japonicus, a thermotolerant strain (group G3: wet weight, 64.22 ± 13.16 g/ind.), which was selected focusing on the performance of thermo-tolerance through three generations for 10 years, and a control group (group C: wet weight, 62.08 ± 12.01 g/ind.) were collected from July to October in a seawater pond in northern China. Differences in relative faecal mass (RFM), relative intestinal mass (RIM), percentage of individuals with faeces (PIWF) and intestinal metabolites were investigated and compared between the two groups. The temperatures of terminating aestivation of the two groups were assessed according to the values of RFM and RIM. A regression analysis showed that the corresponding group G3 temperatures were 0.56–1.34°C and 0.70–1.49°C higher than those of group C when RFM and RIM were 0.01–0.05 and 0.005–0.025 respectively. The PIWF values in group G3 were 11.5%–21.2% higher than that in group C from 20 July to 22 September. Significant differences at the concentrations of 52 metabolites were detected between the two groups, 36 were higher in group G3 and 16 were higher in group C. The concentrations of threitol, 2-methylglutaric acid, N-acetyl-D-galactosamine, N-acetyl-L-leucine, lactose, oxoproline, 2,3-dimethylsuccinic acid and d-glucoheptose were significantly different between groups G3 and C and were considered metabolic markers distinguishing group G3 from group C. Metabolism of A. japonicus in group G3 was more active than that in group C. These results provide new insight for understanding ingestion and intestinal metabolism in the thermotolerant strain of A. japonicus under high summer temperature.
This chapter discusses the achievements realized in the field of toxicology through the use of the nematode Caenorhabditis elegans as an animal model. The entire lifecycle, from an egg to an adult producing more eggs, takes just 3.5 days. The hermaphrodite reproductive system consists of a symmetrically arranged bilobed gonad. The male gonad is a single lobed U-shaped structure. The developmental stages in C. elegans are very short, and its molecular and morphological pathways are well reported. The adult C. elegans has 302 neurons in two independent nervous systems: 282 in the somatic nervous system and 20 in the pharyngeal nervous system. These nervous systems control locomotion, feeding, defecation, reproduction and environmental sensing. Alterations in any of these functions may be indicators of neurotoxicity. Several studies have shown that some reproductive endpoints are controlled by the nervous system. Since the genome was first published, a plethora of molecular techniques have been developed for genetic manipulation and cellular and molecular observation that are particularly valuable for toxicological studies. Transgenic C. elegans have been employed as biomarkers of a variety of environmental exposures and provide valuable insight regarding the molecular bases of cellular stress and toxicity. Light microscopy is another valuable tool for the examination of toxicant effects in C. elegans. A variety of computer-based assays have been developed to assess outcomes of toxicant exposure in C. elegans. Genome-wide screening using C. elegans is a classical approach for studying the functional repercussions of a gene in a given molecular response. Developmental and reproductive toxicity are caused by metals like cadmium, zinc, mercury, lead, magnesium and other metals as well as pesticides.
Usually, stress is considered adverse: too much work load, or, conversely, unemployment; lack of success; unsolved family problems, etc. More scientifically, Selye (1936) discovered in his fundamental study by challenging rats that “if the organism is severely damaged by acute non-specific nocuous agents such as exposure to cold, surgical injury, production of spinal shock (transcision of the cord), excessive muscular exercise, or intoxications with sublethal doses of diverse drugs (adrenaline, atropine, morphine, formaldehyde, etc.), a typical syndrome appears, the symptoms of which are independent of the nature of the damaging agent or the pharmacological type of the drug employed, and represent rather a response to damage as such”.
How do organisms cope with complex environmental stressors? How do they respond to environmental challenges? Transcription is the initial step in gene expression, thus a transcriptional response gives the first indication of cellular mechanisms that are affected by a stressor. As a consequence, it can provide a sensitive starting point to assess ecological stress responses assuming all ecologically relevant effects are indeed accompanied by alterations in gene expression profiles. Ecological transcriptomics is a powerful tool that unravels mechanistic processes, reveals novel modes of action and regulation pathways, and provides the opportunity to get a dynamic picture of biological systems and the ability to comprehensively dissect different states of biological activities in cells, tissues, or whole organisms.
To most biomolecules, elemental oxygen is inert since it usually does not oxidize them without prior activation either inside or outside of organisms. Atmospheric oxygen in its ground state is distinctive among the gaseous elements because it is a bi-radical. This means it possesses two unpaired electrons with parallel spins which make it paramagnetic. In this constitution, it is very unlikely to participate in reactions with organic molecules unless activated.
This chapter presents an overview of crustacean endocrinology. It highlights potential endocrine-mediated effects observed in crustaceans over the last decade, including end points that are reflective of endocrine disruption in crustaceans. The subchronic/chronic standard test protocols that are available for assessment of chemical substances within the major standardization bodies are summarized, as are some complementary genomic tools, which may be used for improved understanding of endocrine disruption in crustaceans. Finally, the chapter summarizes the main findings and presents some final recommendations.
Unionid freshwater mussels are considered the most endangered group of all freshwater organisms. They have a complex life cycle which includes a parasitic phase on a host fish as well as a post-parasitic phase during which the juveniles depend on interstitial stream substratum properties. This contribution highlights the importance of integrative approaches to identifying the critical bottlenecks in the mussel life cycle in order to deduce sound conservation management strategies. The example of the critically endangered European freshwater pearl mussel (Margaritifera margaritifera) which has a lifespan over 100 years and which has lacked recruitment in the majority of its populations for 30-50 years, illustrates how careful management of fish host populations, stream substratum properties and genetic diversity and differentiation need to be integrated for sustainable conservation. Data on European populations of the species indicate that the stream bed properties are likely to be currently the most limiting factor for survival since this habitat has been strongly altered by landuse changes and stream corrections. Evaluations of stream bed restoration measures revealed that the primary concern of restoration should be catchment erosion management. To prevent genetically unique populations from extinction, artificial culturing of such valuable populations can be an emergency measure.
Six lines of flax ( Linum usitatissimum) were derived from six short, early flowering plants that were first-generation progeny of plants grown from seeds treated with 5-azacytidine. The third and fourth progeny generations of the lines, and the F1 and F2 progeny of crosses between three of the lines and their corresponding untreated controls, demonstrated the relatively stable inheritance of the induced phenotypes and provided information on the genetic control of the induced changes. Some reversion of the induced phenotypes was detected, but much of the phenotypic variability observed in the progeny generations could be attributed to segregation. Segregation patterns demonstrated that the early flowering phenotype is deter- mined by the combined effects of 5-azacytidine-induced changes at three (or more) independent loci. Furthermore, the induced form of one locus is dominant or co- dominant, but the induced forms of the other loci are recessive. A working model describes the proposed genetic control, and the significance of 5-azacytidine- induced effects which are recessive is discussed.
1. This review focuses on direct and indirect interactions between dissolved humic substances (HS) and freshwater organisms and presents novel opinions and hypotheses on their ecological significance. Despite their abundance in freshwaters, the role of HS is still inadequately understood. These substances have been considered too large to be taken up by freshwater organisms. On the contrary, here we present evidence that dissolved HS are indeed taken up and interact directly and/or indirectly with freshwater organisms.
2. We show that dissolved HS exert a mild chemical stress upon aquatic organisms in many ways; they induce molecular chaperones (stress shock proteins), induce and modulate biotransformation enzymes and modulate (mainly inhibiting) the photosynthetic release of oxygen by freshwater plants. Furthermore, they produce an oxidative stress, which may lead to membrane oxidation. HS modulate the multixenobiotic resistance activity and probably other membrane‐bound pumps. This property may lead to the increased bioaccumulation of xenobiotic chemicals. Furthermore, they can modulate the numbers of offspring in a nematode and feminise fish and amphibians. The ecological consequences of this potential remain obscure at present. HS also have the potential to act as chemical attractants (as shown with a nematode).
3. In some macrophytes and algae we show that HS interfere with photosynthesis and growth. For instance, the presence of HS suppresses cyanobacteria more than eukaryotic algae. By applying a quantitative structure activity relationship approach, we show that quinones in the HS interfere with photosynthetic electron transport. We show that even Phragmites leachate can act as a kind of phytotoxin. HS also have the potential to suppress fungal growth, as shown with the water mould Saprolegnia parasitica and force the fungus to respond by spore production.
4. In very soft, humic freshwaters, such as the Rio Negro, Brazil, HS stimulate the uptake of essential ions, such as Na and Ca, at extremely low pH (3.5–4.0) and prevent the ionoregulatory disturbance induced by acid waters, thereby enabling fish to survive in these environments.
5. We discuss whether or not HS are directly utilised by aquatic microorganisms or via exoenzymes, which may be washed in from the terrestrial catchment. There is accumulating evidence that the quality of the HS controls microbial growth. In total, net‐heterotrophy may result from HS‐mediated suppression of primary production by the quinone structures and/or from HS‐mediated support of microbial growth. As there is also evidence that HS have the potential to support photoautotrophic growth and suppress microbial growth, the opposite community effect could result. Consequently, dissolved organic carbon (DOC) has to be chemically characterised, rather than simply measuring bulk DOC concentration.
6. In sum, dissolved HS interact with freshwater organisms in a variety of ways in unenriched humic lakes. In addition to the well known effects of HS on light regime, for example, and the direct and indirect supply with carbon (energy), other interactions may be much more subtle. For instance, HS may induce internal biochemical stress defence systems and have the potential to cause acclimatisation and even adaptation. We are just at the beginning of understanding these interactions between dissolved HS and freshwater organisms.
Many aquatic species, including the estuarine fish Fundulus heteroclitus (mummichogs), adapt to local environmental conditions. We conducted studies to evaluate whether highly exposed populations of mummichogs adapt to toxic environmental contaminants. These fish populations are indigenous to an urban estuary contaminated with persistent and bioaccumulative contaminants (dioxin-like compounds, or DLCs) that are particularly toxic to the early development of fish. We conducted laboratory challenge experiments to compare mummichog embryos and larvae from reference sites and this highly contaminated site [New Bedford Harbor (NBH), Massachusetts, USA] for their sensitivity to DLCs. While there was variation in DLC-responsiveness within each group, fish from NBH were profoundly less sensitive to DLCs than reference fish. Specifically, concentrations of DLCs similar to those measured in NBH-collected mummichog eggs were lethal to reference
embryos. Further, DLC-responsiveness was inherited and independent of maternal contaminant contributions. These findings are consistent with the conclusion that DLC contamination in NBH has contributed to the selection of fish that are resistant to the short-term toxic effects of these environmental-contaminant exposures. This adaptation may be a critical mechanism by which fish populations persist in this highly contaminated site. Further evaluation of this ecosystem may provide important information concerning the direct and indirect consequences of this “unnatural” selection.
To more fully assess the toxicity of water-soluble fullerene (nC60), acute toxicity assays were performed on several environmentally relevant species. Included were the freshwater crustaceans Daphnia magna and Hyalella azteca, and a marine harpacticoid copepod, and two fish species, fathead minnow Pimephales promelas and Japanese medaka Oryzias latipes. The latter two species were used to assess sublethal effects of fullerene exposure by also assessing mRNA and protein expression in liver. Because prior studies found that both sonication and using tetrahydrofuran to solubilize fullerene increased the toxicity of nC60, the nC60 used in this study was prepared by stirring. For the invertebrate studies, nC60 could not be prepared at high enough concentration levels to cause 50% mortality (LC50) at 48 or 96 h. The maximum concentrations tested were 35 ppm for freshwater and 22.5 ppm for full-strength (35 ppt) seawater, since at higher concentrations the nC60 precipitated out of solution. Daphnia 21-day exposures resulted in a significant delay in molting and significantly reduced offspring production at 2.5 and 5 ppm nC60, which could possibly produce impacts at the population-level. For the fish, we found that neither the mRNA nor protein-expression levels of cytochrome P450 isozymes CYP1A, CYP2K1 and CYP2M1 were changed. The peroxisomal lipid transport protein PMP70 was significantly reduced in fathead minnow, but not medaka, indicating potential changes in acyl-CoA pathways.
Microarrays measure the binding of nucleotide sequences to a set of sequence specific probes. This information is combined with annotation specifying the relationship between probes and targets and used to make inferences about transcript- and, ultimately, gene expression. In some situations, a probe is capable of hybridizing to more than one transcript, in others, multiple probes can target a single sequence. These 'multiply targeted' probes can result in non-independence between measured expression levels.
An analysis of these relationships for Affymetrix arrays considered both the extent and influence of exact matches between probe and transcript sequences. For the popular HGU133A array, approximately half of the probesets were found to interact in this way. Both real and simulated expression datasets were used to examine how these effects influenced the expression signal. It was found not only to lead to increased signal strength for the affected probesets, but the major effect is to significantly increase their correlation, even in situations when only a single probe from a probeset was involved. By building a network of probe-probeset-transcript relationships, it is possible to identify families of interacting probesets. More than 10% of the families contain members annotated to different genes or even different Unigene clusters. Within a family, a mixture of genuine biological and artefactual correlations can occur.
Multiple targeting is not only prevalent, but also significant. The ability of probesets to hybridize to more than one gene product can lead to false positives when analysing gene expression. Comprehensive annotation describing multiple targeting is required when interpreting array data.
New methods are needed for research into non-model organisms, to monitor the effects of toxic disruption at both the molecular and functional organism level. We exposed earthworms (Lumbricus rubellus Hoffmeister) to sub-lethal levels of copper (10-480 mg/kg soil) for 70 days as a real-world situation, and monitored both molecular (cDNA transcript microarrays and nuclear magnetic resonance-based metabolic profiling: metabolomics) and ecological/functional endpoints (reproduction rate and weight change, which have direct relevance to population-level impacts).
Both of the molecular endpoints, metabolomics and transcriptomics, were highly sensitive, with clear copper-induced differences even at levels below those that caused a reduction in reproductive parameters. The microarray and metabolomic data provided evidence that the copper exposure led to a disruption of energy metabolism: transcripts of enzymes from oxidative phosphorylation were significantly over-represented, and increases in transcripts of carbohydrate metabolising enzymes (maltase-glucoamylase, mannosidase) had corresponding decreases in small-molecule metabolites (glucose, mannose). Treating both enzymes and metabolites as functional cohorts led to clear inferences about changes in energetic metabolism (carbohydrate use and oxidative phosphorylation), which would not have been possible by taking a 'biomarker' approach to data analysis.
Multiple post-genomic techniques can be combined to provide mechanistic information about the toxic effects of chemical contaminants, even for non-model organisms with few additional mechanistic toxicological data. With 70-day no-observed-effect and lowest-observed-effect concentrations (NOEC and LOEC) of 10 and 40 mg kg-1 for metabolomic and microarray profiles, copper is shown to interfere with energy metabolism in an important soil organism at an ecologically and functionally relevant level.
Natural contamination and anthropogenic pollution of soils are likely to be major determinants of functioning and survival of keystone invertebrate taxa. Soil animals will have both evolutionary adaptation and genetically programmed responses to these toxic chemicals, but mechanistic understanding of such is sparse. The clitellate annelid Lumbricus rubellus is a model organism for soil health testing, but genetic data have been lacking.
We generated a 17,000 sequence expressed sequence tag dataset, defining ~8,100 different putative genes, and built an 8,000-element transcriptome microarray for L. rubellus. Strikingly, less than half the putative genes (43%) were assigned annotations from the gene ontology (GO) system; this reflects the phylogenetic uniqueness of earthworms compared to the well-annotated model animals. The microarray was used to identify adult- and juvenile-specific transcript profiles in untreated animals and to determine dose-response transcription profiles following exposure to three xenobiotics from different chemical classes: inorganic (the metal cadmium), organic (the polycyclic aromatic hydrocarbon fluoranthene), and agrochemical (the herbicide atrazine). Analysis of these profiles revealed compound-specific fingerprints which identify the molecular responses of this annelid to each contaminant. The data and analyses are available in an integrated database, LumbriBASE.
L. rubellus has a complex response to contaminant exposure, but this can be efficiently analysed using molecular methods, revealing unique response profiles for different classes of effector. These profiles may assist in the development of novel monitoring or bioremediation protocols, as well as in understanding the ecosystem effects of exposure.
Microarray analysis has become a widely used tool for the generation of gene expression data on a genomic scale. Although many significant results have been derived from microarray studies, one limitation has been the lack of standards for presenting and exchanging such data. Here we present a proposal, the Minimum Information About a Microarray Experiment (MIAME), that describes the minimum information required to ensure that microarray data can be easily interpreted and that results derived from its analysis can be independently verified. The ultimate goal of this work is to establish a standard for recording and reporting microarray-based gene expression data, which will in turn facilitate the establishment of databases and public repositories and enable the development of data analysis tools. With respect to MIAME, we concentrate on defining the content and structure of the necessary information rather than the technical format for capturing it.
Microarray analysis has become a widely used tool for the generation of gene expression data on a genomic scale. Although many significant results have been derived from microarray studies, one limitation has been the lack of standards for presenting and exchanging such data. Here we present a proposal, the Minimum Information About a Microarray Experiment (MIAME), that describes the minimum information required to ensure that microarray data can be easily interpreted and that results derived from its analysis can be independently verified. The ultimate goal of this work is to establish a standard for recording and reporting microarray-based gene expression data, which will in turn facilitate the establishment of databases and public repositories and enable the development of data analysis tools. With respect to MIAME, we concentrate on defining the content and structure of the necessary information rather than the technical format for capturing it.
Dissolved humic substances (HSs) are taken up by organisms and interact on various molecular and biochemical levels. With two examples, we show that HSs facilitate fish life in low pH and low calcium waters and promote longevity by mild chemical stress. We re-visit recent papers on fish adaptation in the Amazonian Rio Negro and re-interpret the results on the basis of the recent finding of gene control in the nematode Caenorhabditis elegans. In the nematode, several genes are up-regulated. This may also account for the maintenance of fish life in the Rio Negro. Exposure to HSs exerts mild chemical stress on the exposed organisms and deprives them of energy; however, the nematode C. elegans actively looks for such environments. With life-table and DNA gene filter studies we show that HSs may cause longevity and multiple stress resistance. Furthermore, we propose likely structures of the HSs responsible for these effects.
The heat-shock protein Hsp90 supports diverse but specific signal transducers and lies at the interface of several developmental pathways. We report here that when Drosophila Hsp90 is mutant or pharmacologically impaired, phenotypic variation affecting nearly any adult structure is produced, with specific variants depending on the genetic background and occurring both in laboratory strains and in wild populations. Multiple, previously silent, genetic determinants produced these variants and, when enriched by selection, they rapidly became independent of the Hsp90 mutation. Therefore, widespread variation affecting morphogenic pathways exists in nature, but is usually silent; Hsp90 buffers this variation, allowing it to accumulate under neutral conditions. When Hsp90 buffering is compromised, for example by temperature, cryptic variants are expressed and selection can lead to the continued expression of these traits, even when Hsp90 function is restored. This provides a plausible mechanism for promoting evolutionary change in otherwise entrenched developmental processes.
The rapid discovery of sequence information from the Human Genome Project has exponentially increased the amount of data that can be retrieved from biomedical experiments. Gene expression profiling, through the use of microarray technology, is rapidly contributing to an improved understanding of global, coordinated cellular events in a variety of paradigms. In the field of toxicology, the potential application of toxicogenomics to indicate the toxicity of unknown compounds has been suggested but remains largely unsubstantiated to date. A major supposition of toxicogenomics is that global changes in the expression of individual mRNAs (i.e., the transcriptional responses of cells to toxicants) will be sufficiently distinct, robust, and reproducible to allow discrimination of toxicants from different classes. Definitive demonstration is still lacking for such specific "genetic fingerprints," as opposed to nonspecific general stress responses that may be indistinguishable between compounds and therefore not suitable as probes of toxic mechanisms. The present studies demonstrate a general application of toxicogenomics that distinguishes two mechanistically unrelated classes of toxicants (cytotoxic anti-inflammatory drugs and DNA-damaging agents) based solely upon a cluster-type analysis of genes differentially induced or repressed in cultured cells during exposure to these compounds. Initial comparisons of the expression patterns for 100 toxic compounds, using all approximately 250 genes on a DNA microarray ( approximately 2.5 million data points), failed to discriminate between toxicant classes. A major obstacle encountered in these studies was the lack of reproducible gene responses, presumably due to biological variability and technological limitations. Thus multiple replicate observations for the prototypical DNA damaging agent, cisplatin, and the non-steroidal anti-inflammatory drugs (NSAIDs) diflunisal and flufenamic acid were made, and a subset of genes yielding reproducible inductions/repressions was selected for comparison. Many of the "fingerprint genes" identified in these studies were consistent with previous observations reported in the literature (e. g., the well-characterized induction by cisplatin of p53-regulated transcripts such as p21(waf1/cip1) and PCNA [proliferating cell nuclear antigen]). These gene subsets not only discriminated among the three compounds in the learning set but also showed predictive value for the rest of the database ( approximately 100 compounds of various toxic mechanisms). Further refinement of the clustering strategy, using a computer-based optimization algorithm, yielded even better results and demonstrated that genes that ultimately best discriminated between DNA damage and NSAIDs were involved in such diverse processes as DNA repair, xenobiotic metabolism, transcriptional activation, structural maintenance, cell cycle control, signal transduction, and apoptosis. The determination of genes whose responses appropriately group and dissociate anti-inflammatory versus DNA-damaging agents provides an initial paradigm upon which to build for future, higher throughput-based identification of toxic compounds using gene expression patterns alone.
Heat-shock protein 90 (Hsp90) chaperones the maturation of many regulatory proteins and, in the fruitfly Drosophila melanogaster, buffers genetic variation in morphogenetic pathways. Levels and patterns of genetic variation differ greatly between obligatorily outbreeding species such as fruitflies and self-fertilizing species such as the plant Arabidopsis thaliana. Also, plant development is more plastic, being coupled to environmental cues. Here we report that, in Arabidopsis accessions and recombinant inbred lines, reducing Hsp90 function produces an array of morphological phenotypes, which are dependent on underlying genetic variation. The strength and breadth of Hsp90's effects on the buffering and release of genetic variation suggests it may have an impact on evolutionary processes. We also show that Hsp90 influences morphogenetic responses to environmental cues and buffers normal development from destabilizing effects of stochastic processes. Manipulating Hsp90's buffering capacity offers a tool for harnessing cryptic genetic variation and for elucidating the interplay between genotypes, environments and stochastic events in the determination of phenotype.
Gene expression of Escherichia coli cells exposed to seawater for 20 h was compared to that of exponentially growing cells (mops-glucose 0.2%) using DNA microarray technology. The expression of most (ca. 3,000) of the 4,228 open reading frames on the microarray remained unchanged; the relative expression of about 320 genes decreased in seawater, whereas that of ca. one fourth (937) increased. Clearly coherent expression patterns were observed for several functional gene groups. Induced genes were numerous in groups specifying the degradation of small molecules (carbon compounds, amino acids and fatty acids), energy metabolism (aerobic and anaerobic respiration, pyruvate dehydrogenase and TCA cycle), chemotaxis and mobility, flagella biosynthesis, surface structures and phage related functions. Repressed genes were clustered in two groups, cell division and nucleotides biosynthesis, indicating a cessation of growth.
Morphological alterations have been shown to occur in Drosophila melanogaster when function of Hsp90 (heat shock 90-kDa protein 1alpha, encoded by Hsp83) is compromised during development. Genetic selection maintains the altered phenotypes in subsequent generations. Recent experiments have shown, however, that phenotypic variation still occurs in nearly isogenic recombinant inbred strains of Arabidopsis thaliana. Using a sensitized isogenic D. melanogaster strain, iso-Kr(If-1), we confirm this finding and present evidence supporting an epigenetic mechanism for Hsp90's capacitor function, whereby reduced activity of Hsp90 induces a heritably altered chromatin state. The altered chromatin state is evidenced by ectopic expression of the morphogen wingless in eye imaginal discs and a corresponding abnormal eye phenotype, both of which are epigenetically heritable in subsequent generations, even when function of Hsp90 is restored. Mutations in nine different genes of the trithorax group that encode chromatin-remodeling proteins also induce the abnormal phenotype. These findings suggest that Hsp90 acts as a capacitor for morphological evolution through epigenetic and genetic mechanisms.
The National Center for Toxicogenomics is developing the first public toxicogenomics knowledge base that combines molecular expression data sets from transcriptomics, proteomics, metabonomics, and conventional toxicology with metabolic, toxicologcal pathway, and gene regulatory network information relevant to environmental toxicology and human disease. It is called the Chemical Effects in Biological Systems (CEBS) knowledge base and is designed to meet the information needs of "systems toxicology," involving the study of perturbation by chemicals and stressors, monitoring changes in molecular expression and conventional toxicological parameters, and iteratively integrating biological response data to describe the functioning organism. Based upon functional genomics approaches used successfully in analyzing yeast gene expression data sets, relational and descriptive compendia will be assembled for toxicologically important genes, groups of genes, single nucleotide polymorphisms (SNPs), and mutant and knockout phenotypes. CEBS data sets will be fully documented in the experimental protocol and therefore searchable by compound, structure, toxicity end point, pathology and point, gene, gene group, SNP, pathway, and network as a function of dose, time, and the phenotype of the target tissue. A knowledge base is being developed by assimilating toxicological, biological, and chemical information from multiple public domain databases and by progressively refining that information about gene, protein, and metabolite expression for classes of chemicals and their biological effects in various species. By analogy to the GenBank database for genome sequences, researchers will globally query (or BLAST) CEBS using a transcriptome of a tissue of interest (or a list of outliers) to have the knowledge base return information on genes, groups of genes, metabolic and toxicological pathways, and contextually associated phenotypic information for compounds that display similar response profiles. With high-quality data content, CEBS will ultimately become a resource to support hypothesis-driven and discovery research that contributes effectively to drug safety and the improvement of risk assessments for chemicals in the environment. The CEBS development effort will span a decade or more.
Serial analysis of gene expression was used to profile transcript levels in Arabidopsis roots and assess their responses to 2,4,6-trinitrotoluene (TNT) exposure. SAGE libraries representing control and TNT-exposed seedling root transcripts were constructed, and each was sequenced to a depth of roughly 32,000 tags. More than 19,000 unique tags were identified overall. The second most highly induced tag (27-fold increase) represented a glutathione S-transferase. Cytochrome P450 enzymes, as well as an ABC transporter and a probable nitroreductase, were highly induced by TNT exposure. Analyses also revealed an oxidative stress response upon TNT exposure. Although some increases were anticipated in light of current models for xenobiotic metabolism in plants, evidence for unsuspected conjugation pathways was also noted. Identifying transcriptome-level responses to TNT exposure will better define the metabolic pathways plants use to detoxify this xenobiotic compound, which should help improve phytoremediation strategies directed at TNT and other nitroaromatic compounds.
Novel molecular based methods are being developed to study changes in gene expression in wildlife exposed to anthropogenic chemicals. Gene arrays, in particular, are useful tools that can be used to simultaneously monitor hundreds to thousands of genes within a single experiment, giving an investigator the ability to determine how exposure affects multiple metabolic pathways. These methods are thought to be both sensitive and able to reveal biochemical mechanisms of action. A largemouth bass (LMB) array containing 132 genes has been designed to study the impact of gene expression in male fish exposed to 17-beta estradiol or to the compounds 4-nonylphenol (4-NP) or 1,1-dichloro-2, 2-bis (p-chlorophenyl) ethylene (p,p'-DDE). The results of these experiments demonstrate distinct gene expression patterns in LMB exposed to these compounds.
The Microarray Gene Expression Data Society believe that the time is right for journals to require that microarray data be deposited in public repositories, as a condition for publication.
Understanding the function of detoxifying enzymes in plants toward xenobiotics is of major importance for phytoremediation applications. In this work, Arabidopsis (Arabidopsis thaliana; ecotype Columbia) seedlings were exposed to 0.6 mm acetochlor (AOC), 2 mm metolachlor (MOC), 0.6 mm 2,4,6-trinitrotoluene (TNT), and 0.3 mm hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). In vivo glutathione (GSH) conjugation reactions of AOC, MOC, RDX, and TNT were studied in root cells using a multiphoton microscope. In situ labeling with monochlorobimane, used as a competitive compound for conjugation reactions with GSH, confirmed that AOC and MOC are conjugated in Arabidopsis cells. Reverse transcription-PCR established the expression profile of glutathione S-transferases (GSTs) and nitroreductases enzymes. Genes selected for this study were AtGSTF2, AtGSTU1, AtGSTU24, and two isoforms of 12-oxophytodienoate reductase (OPR1 and OPR2). The five transcripts tested were induced by all treatments, but RDX resulted in low induction. The mRNA level of AtGSTU24 showed substantial increase for all chemicals (23-fold induction for AOC, 18-fold for MOC, 5-fold for RDX, and 40-fold for TNT). It appears that GSTs are also involved in the conjugation reactions with metabolites of TNT, and to a lesser extent with RDX. Results indicate that OPR2 is involved in plant metabolism of TNT (11-fold induction), and in oxidative stress when exposed to AOC (7-fold), MOC (9-fold), and RDX (2-fold). This study comprises gene expression analysis of Arabidopsis exposed to RDX and AOC, which are considered significant environmental contaminants, and demonstrates the importance of microscopy methods for phytoremediation investigations.
Background: Microarrays measure the binding of nucleotide sequences to a set of sequence specific probes. This information is combined with annotation specifying the relationship between probes and targets and used to make inferences about transcript- and, ultimately, gene expression. In some situations, a probe is capable of hybridizing to more than one transcript, in others, multiple probes can target a single sequence. These 'multiply targeted' probes can result in nonindependence between measured expression levels. Results: An analysis of these relationships for Affymetrix arrays considered both the extent and influence of exact matches between probe and transcript sequences. For the popular HGU133A array, approximately half of the probesets were found to interact in this way. Both real and simulated expression datasets were used to examine how these effects influenced the expression signal. It was found not only to lead to increased signal strength for the affected probesets, but the major effect is to significantly increase their correlation, even in situations when only a single probe from a probeset was involved. By building a network of probe-probeset-transcript relationships, it is possible to identify families of interacting probesets. More than 10% of the families contain members annotated to different genes or even different Unigene clusters. Within a family, a mixture of genuine biological and artefactual correlations can occur. Conclusion: Multiple targeting is not only prevalent, but also significant. The ability of probesets to hybridize to more than one gene product can lead to false positives when analysing gene expression. Comprehensive annotation describing multiple targeting is required when interpreting array data.
We have mapped and quantified mouse transcriptomes by deeply sequencing them and recording how frequently each gene is represented in the sequence sample ( RNA - Seq ). This provides a digital measure of the presence and prevalence of transcripts from known ...
Arabidopsis thaliana root transcriptome responses to the munition, hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), were assessed using serial analysis of gene expression (SAGE). Sequencing of SAGE libraries from control and RDX-exposed root tissues revealed induction of genes known to respond to a variety of general stresses. Among the highly induced genes were several encoding molecular chaperones and transcription factors as well as vacuolar proteins and peroxidases. Strongly repressed transcripts included ones encoding ribosomal proteins, a cyclophilin, a katanin, and a peroxidase. Comparison of the transcriptional profile for the RDX response to a profile previously described for Arabidopsis roots exposed to trinitrotoluene (TNT) revealed significant differences in the inferred gene expression patterns. This suggests that Arabidopsis employs drastically different mechanisms for coping with these two compounds. With respect to the goal of engineering plants to better tolerate and degrade explosives at contaminated sites, these results suggest that enhancement of different genes and metabolic pathways may be required to deal effectively with each type of explosive. This has ramifications for phytoremediation efforts since many contaminated sites harbor both compounds.
The rapid discovery of sequence information from the Human Genome Project has exponentially increased the amount of data that can be retrieved from biomedical experiments. Gene expression profiling, through the use of microarray technology, is rapidly contributing to an improved understanding of global, coordinated cellular events in a variety of paradigms. In the field of toxicology, the potential application of toxicogenomics to indicate the toxicity of unknown compounds has been suggested but remains largely unsubstantiated to date. A major supposition of toxicogenomics is that global changes in the expression of individual mRNAs (i.e., the transcriptional responses of cells to toxicants) will be sufficiently distinct, robust, and reproducible to allow discrimination of toxicants from different classes. Definitive demonstration is still lacking for such specific “genetic fingerprints,” as opposed to nonspecific general stress responses that may be indistinguishable between compounds and therefore not suitable as probes of toxic mechanisms. The present studies demonstrate a general application of toxicogenomics that distinguishes two mechanistically unrelated classes of toxicants (cytotoxic anti-inflammatory drugs and DNA-damaging agents) based solely upon a cluster-type analysis of genes differentially induced or repressed in cultured cells during exposure to these compounds. Initial comparisons of the expression patterns for 100 toxic compounds, using all ; 250 genes on a DNA microarray (; 2.5 million data points), failed to discriminate between toxicant classes. A major obstacle encountered in these studies was the lack of reproducible gene responses, presumably due to biological variability and technological limitations. Thus multiple replicate observations for the prototypical DNA damaging agent, cisplatin, and the non-steroidal anti-inflammatory drugs (NSAIDs) diflunisal and flufenamic acid were made, and a subset of genes yielding reproducible inductions/repressions was selected for comparison. Many of the “fingerprint genes” identified in these studies were consistent with previous observations reported in the literature (e.g., the well-characterized induction by cisplatin of p53-regulated transcripts such as p21 waf1/cip1 and PCNA [proliferating cell nuclear antigen]). These gene subsets not only discriminated among the three compounds in the learning set but also showed predictive value for the rest of the database (; 100 compounds of various toxic mechanisms). Further refinement of the clustering strategy, using a computer-based optimization algorithm, yielded even better results and demonstrated that genes that ultimately best discriminated between DNA damage and NSAIDs were involved in such diverse processes as DNA repair, xenobiotic metabolism, transcriptional activation, structural main
In plants, naturally occurring methylation of genes can affect the level of gene expression. Variation among individuals in the degree of methylation of a gene, termed epialleles, produces novel phenotypes that are heritable across generations. To date, ecologically important genes with methylated epialleles have been found to affect floral shape, vegetative and seed pigmentation, pathogen resistance and development in plants. Currently, the extent to which epiallelic variation is an important common contributor to phenotypic variation in natural plant populations and its fitness consequences are not known. Because epiallele phenotypes can have identical underlying DNA sequences, response to selection on these phenotypes is likely to differ from expectations based on traditional models of microevolution. Research is needed to understand the role of epialleles in natural plant populations. Recent advances in molecular genetic techniques could enable population biologists to screen for epiallelic variants within plant populations and disentangle epigenetic from more standard genetic sources of phenotypic variance, such as additive genetic variance, dominance variance, epistasis and maternal genetic effects.
Cited By (since 1996):24, Export Date: 28 October 2013, Source: Scopus
Cited By (since 1996):24, Export Date: 28 October 2013, Source: Scopus
1. We investigated the effect of refractory dissolved organic matter (refractory DOM: fulvic acids (FAs) and ultrafiltrates (UFs)), isolated from five different sources, on the reproduction of the bacterivorous nematode Caenorhabditis elegans . Nematodes were exposed to DOM (0.5–64 mg L−1 dissolved organic carbon) during a whole life cycle (72 h). At the end of the test, the number of offspring per worm was determined.
2. We also studied the effect of refractory DOM on abundance, cell size, and activity of the bacteria ( Escherichia coli ) that were used as a food source for the nematodes, to assess possible indirect effects of DOM via the food organisms.
3. The effects of DOM on the reproduction of C. elegans varied, depending on the origin and concentration of DOM. FAs isolated from a soil leachate and from the effluent of a waste water plant, as well as UFs from a humic lake and from a marsh, stimulated the reproduction of C. elegans. FAs from ground water had no effect, while FAs from a humic lake inhibited the reproduction of the nematodes. All effects occurred at ecologically relevant DOM concentrations and showed clear dose–response relationships.
4. Neither bacterial abundance nor cell size were influenced by refractory DOM. Bacterial activity was unaffected by four types of DOM. Only FAs from the humic lake caused a significant decrease in bacterial activity over 72 h.
5. The negative effect of FAs from the humic lake on nematode reproduction may be a consequence of a lower bacterial activity in this treatment. The positive effects of refractory DOM, however, could not be related to bacterial parameters. Therefore, we assume that the DOM directly influenced the reproduction of C. elegans . We speculate that refractory DOM can potentially be an additional carbon source or a source of trace nutrients influencing the reproduction of C. elegans . Adsorption of refractory DOM on bacterial cells, serving as food for the nematodes, may have been an important factor for the availability of DOM for C. elegans .
2,4,6-trinitrotoluene (TNT) is a nitro-substituted xenobiotic explosive that is toxic to plants and animals. Plants absorb and metabolize TNT, but the pathways are uncertain and plant responses at the molecular level have not been adequately characterized. We analyzed gene expression in response to relatively long-term exposure to TNT at low and high concentration in Arabidopsis through the use of cDNA microarrays. Arabidopsis seedlings were grown on media containing 1 and 10 μM TNT, concentrations that were empirically determined by plant growth analysis. Microarray analysis revealed that a total of 52 genes were significantly upregulated, and 47 genes were downregulated in response to TNT at a 1.7-fold cut-off for differential gene expression. A substantial number of these genes have predicted functions in cell defense and detoxification. Conserved motifs were discovered in the promoter region of co-regulated genes, some of which are potentially novel cis-regulatory elements. With follow-up real time RT-PCR, we confirmed findings from the microarray experiments and examined the regulation of selected genes to two other xenobiotic substances: the explosive RDX and thiodiglycol. Results showed that increased transcription of At5g61600 encoding for a DNA-binding protein in shoots is specific to TNT and increased transcription of At5g42530 encoding for an unknown protein in shoots is specific to both TNT and RDX.
High-aspect rotating vessels (HARVs) are used to study the effects low-shear modeled microgravity (LSMMG) on bacterial gene expression. LSMMG is generated by orienting HARVs with the axis of rotation perpendicular to the gravity vector while gravitational controls are oriented with the axis of rotation parallel to the gravity vector. Microarray analysis was performed on Streptococcus pneumoniae TIGR4 grown in HARVs under three conditions (LSMMG, 1×g, and static) to determine if global transcriptional activity is altered between different gravitational controls and LSMMG. Results revealed 101 differentially expressed genes under static conditions compared to 1×g, 46 genes between 1×g and LSMMG, and nine genes between static and LSMMG. Hierarchical cluster analysis revealed 15 genes exhibiting similar expression patterns under static conditions compared to 1×g. These results indicate that rotation, in addition to low-shear forces, might contribute to bacterial adaptation to the LSMMG.
Keywords: Streptococcus pneumoniae; Microgravity; Microarrays; High-aspect rotating vessels; Virulence
Article Outline
1. Introduction
2. Materials and methods
2.1. Culture and counting of cells in HARVs
2.2. Microarrays
2.3. Real-time reverse transcriptase polymerase chain reaction (RT-PCR)
3. Results
3.1. Gene expression profile of S. pneumoniae under 1×g vs static conditions using microarrays
3.2. Confirmation of the regulation of S. pneumoniae genes by real-time RT-PCR
3.3. Hierarchical clustering reveals a pattern of gene expression for S. pneumoniae grown under 1×g vs static conditions
3.4. Discussion
Acknowledgements
The micronutrient zinc has an essential role in physiological and metabolic processes in plants as a cofactor or structural element in 300 catalytic and noncatalytic proteins, but it is very toxic when available in elevated amounts. Plants tightly regulate their internal zinc concentrations in a process called zinc homeostasis. The exceptional zinc hyperaccumulator species Thlaspi caerulescens can accumulate up to 3% of zinc, but also high amounts of nickel and cadmium, without any sign of toxicity. This should have drastic effects on the zinc homeostasis mechanism. We examined in detail the transcription profiles of roots of Arabidopsis thaliana and T. caerulescens plants grown under deficient, sufficient, and excess supply of zinc. A total of 608 zinc-responsive genes with at least a 3-fold difference in expression level were detected in A. thaliana and 352 in T. caerulescens in response to changes in zinc supply. Only 14% of these genes were also zinc responsive in A. thaliana. When comparing A. thaliana with T. caerulescens at each zinc exposure, more than 2,200 genes were significantly differentially expressed (5-fold and false discovery rate < 0.05). While a large fraction of these genes are of yet unknown function, many genes with a different expression between A. thaliana and T. caerulescens appear to function in metal homeostasis, in abiotic stress response, and in lignin biosynthesis. The high expression of lignin biosynthesis genes corresponds to the deposition of lignin in the endodermis, of which there are two layers in T. caerulescens roots and only one in A. thaliana
We investigated plant cell division by testing for the presence and involvement in progress through the division cycle of the protein p34cdc2, a key participant in division control in other eukaryotes. A protein of the same m(r) 34,000 has structural similarity indicated by its reaction with three sorts of antibody raised against (1) cell division-specific regions within a 16-amino acid internal sequence that is perfectly conserved in p34cdc2 from all known sources, (2) the carboxy-terminal 127 amino acids of human p34cdc2 linked to beta-galactosidase, and (3) whole p34cdc2 of fission yeast. Participation of p34 in the division cycle of the green plant Chlamydomonas is indicated by phosphorylation of the protein only in proliferating cells. There is a consistent fivefold increase relative to other proteins when cells become committed to division and a maximum of phosphorylation at the time of nuclear division under conditions that alter by twofold the time of these events. A p34 protein is detectable in oats and Arabidopsis and in remote taxa, including red and brown algae. We conclude that the plant kingdom shares a division control involving p34cdc2 that was probably established in the common ancestral eukaryote prior to divergence of any of the major eukaryote taxa.
The present article represents a comprehensive effort to assess the hypothesis that hormesis is a highly generalizable biological phenomenon independent of environmental stressor, biological endpoint, and experimental model system. The evaluative methodology and complementary approaches employed to assess this question are (1) evolutionary biology-based theoretical paradigm; (2) evaluation of > 20,000 toxicology articles using a priori entry and evaluative criteria; (3) evaluation of 17 large-scale studies each providing data on numerous agents tested in the same experimental model by the same research team; (4) the assimilation of experimental pharmacological data on 24 receptor systems in which biphasic dose responses have been established reproducibly along with hormetic mechanism elucidation; and (5) assessment of the original hormesis database with 1600 dose-response relationships demonstrating evidence consistent with the hormesis hypothesis. The complementary approaches for assessing hormesis provided strong support for its credibility as a central biological theory based on its high frequency of occurrence and quantitative features of expression within microbe, plant, and invertebrate and vertebrate animal systems. The findings suggest that hormetic effects represent evolutionary-based adaptive responses to environmentally induced disruptions in homeostasis. Such adaptive responses, which are incorporated into organismal integrative physiological systems and now clarified at the mechanistic level for more than two dozen receptor systems, provide a cogent basis for the application of hormetic mechanisms in the elucidation of fundamental evolutionary-based biological processes and in the development of novel clinical modalities.
In order to assist in the identification of possible endocrine disrupting chemicals (EDC) in groundwater, we are developing Caenorhabolitis elegans as a high throughput bioassay system in which responses to EDC may be detected by gene expression using DNA microarray analysis. As a first step we examined gene expression patterns and vitellogenin responses of this organism to vertebrate steroids, in liquid culture. Western blotting showed the expected number and size of vitellogenin translation products after estrogen exposure. At 10(-9) M, vitellogenin decreased, but at 10(-7) and 10(-5), vitellogenin was increased. Testosterone (10(-5) M) increased the synthesis of vitellogenin, but progesterone-treated cultures (10(-5) M) had less vitellogenin. Using DNA microarray analysis, we examined the pattern of gene expression after progesterone (10(-5), 10(-7), and 10(-9) M), estrogen (10(-5) M), and testosterone (10(-9) M) exposure, with special attention to the traditional biomarker genes used in environmental studies [vitellogenin, cytochrome P450 (CYP), glutathione s-transferase (GST), metallothionein (MT), and heat shock proteins (HSP)]. GST and P450 genes were affected by estrogen (10(-5) M) and progesterone (10(-5) and 10(-7) M) treatments. For vitellogenin genes, estrogen treatment (10(-5) M) caused overexpression of the vit-2 and vit-6 genes (2.68 and 3.25 times, respectively). After progesterone treatment (10(-7) M), the vit-5 and vit-6 were down-regulated and vit-1 up-regulated (3.59-fold). Concentrations of testosterone and progesterone at 10(-9) M did not influence the expression of the vit, CYP, or GST genes. Although the analysis is incomplete, and low doses and combinations of EDC need to be tested, these preliminary results indicate C. elegans may be a useful laboratory and field model for screening EDC.
Toxicogenomics is a term that represents the merging of toxicology with novel genomics techniques. Data generated in the new‐age era of toxicology is relatively complex, requires new bioinformatics tools for adequate interpretation, and allows for the rapid generation of testable hypotheses. Hazard identification and risk assessment processes will advance from the use of genomics techniques, which will lead to greater understanding of mechanism(s) of action of toxicants, development of novel biomarkers of exposure and effect, and better identification of sensitive subpopulations. © 2001 John Wiley & Sons, Inc. * This article is a US Government work and, as such, is in the public domain in the United States of America.
J Biochem Mol Toxicol 15:231–242, 2001
Traditionally, reactive oxygen intermediates (ROIs) were considered to be toxic by-products of aerobic metabolism, which were disposed of using antioxidants. However, in recent years, it has become apparent that plants actively produce ROIs as signaling molecules to control processes such as programmed cell death, abiotic stress responses, pathogen defense and systemic signaling. Recent advances including microarray studies and the development of mutants with altered ROI-scavenging mechanisms provide new insights into how the steady-state level of ROIs are controlled in cells. In addition, key steps of the signal transduction pathway that senses ROIs in plants have been identified. These raise several intriguing questions about the relationships between ROI signaling, ROI stress and the production and scavenging of ROIs in the different cellular compartments.
Killifish (Fundulus heteroclitus) from a highly contaminated site on the Elizabeth River are resistant to the acute toxicity and the cytochrome P4501A (CYP1A)-inducing activity of both the sediments from the site and chemically pure polycyclic aromatic hydrocarbons (PAHs). These effects are highly heritable for one generation, but heritable to a lesser degree by subsequent generations, in clean conditions in the laboratory. We show that offspring of this population of Elizabeth River killifish are also resistant to the teratogenicity and P4501A-inducing activity of PCB congener 126, a prototypical coplanar halogenated aromatic hydrocarbon (HAH). Furthermore, the pattern of greater resistance to acute toxicity and P4501A-inducing activity in the first generation and less in subsequent generations is also observed upon exposure to PCB-126.
A variety of anthropogenic chemicals are capable of binding to the estrogen receptor of vertebrate species. Binding of these compounds can interfere with homeostasis by disrupting normal gene expression patterns. The purpose of this study was to investigate the feasibility of applying array technology as a monitoring tool for detecting the presence and distribution of estrogenic compounds in coastal habitats using sheepshead minnows as our model. cDNA clones that were isolated from differential display, including vitellogenin alpha and beta, vitelline envelope protein (ZP2), and transferrin, among others, were spotted on the macroarray. The results of these experiments demonstrate a characteristic expression pattern of estrogen responsive genes in sheepshead minnows exposed to 17 beta-estradiol (E2).
The purpose of this study was to determine the specific expression profile of 132 genes, some of which are estrogen responsive, in largemouth bass (LMB) following exposure to estradiol (E(2)), or to two hormonally active agents, 4-nonylphenol (4-NP) and 1,1-dichloro-2, 2-bis (p-chlorophenyl) ethylene (p,p'-DDE), using gene array technology. The results of these experiments show that LMB exposed to E(2) and 4-NP had similar, but not identical genetic signatures for the genes examined, some of which are known to be estrogen-responsive genes. The differences suggest that 4-NP may have additional modes of action that are independent of the estrogen receptor (ER). We have also shown that exposure of male LMB to p,p'-DDE results in an increase in some estrogen-responsive genes. But in female LMB, the observed changes were a down-regulation of the normally up-regulated estrogen responsive genes. Other genes were also down-regulated. These results suggest that p,p'-DDE may affect regulation of genes differently in male and female LMB. This study further suggests that gene arrays have the potential to map out the gene activation pathways of hormonally active compounds.
Suppression subtractive hybridisation (SSH) was used to generate cDNA libraries representing genes differentially expressed in response to ethynyl oestradiol (EE2) exposure in liver from male plaice (Pleuronectes platessa) previously analysed for vitellogenin (VTG) induction. Characterisation of the cDNA clones identified many as VTG (2 genes) and zona radiata proteins (ZRP) (3 genes), but 40 encoded other proteins, with more than half cryptic. Further analysis identified 85 non-redundant clones suitable for array on nylon membrane. Radiolabelled cDNAs were prepared from hepatic mRNA from EE2 treated plaice (0 and 21 days) and hybridised with the arrayed clones. Analysis of the data showed that 11/17 novel, 21/22 VTG, 13/14 ZRP, 2/2 liver aspartic proteinase (LAP) and 8/10 other mRNAs were up-regulated by EE2 exposure.
Humic substances comprise the majority of organic matter in freshwater ecosystems and were thought to be inert or refractory, except for photolytic degradation. However, evidence is increasing that humic substances interact with aquatic organisms similarly to weak anthropogenic chemicals with nonspecific and specific effects. One specific effect is a hormonelike effect, namely, modulation of the number of offspring, which was first described with the nematode Caenorhabditis elegans. Yet a hormonelike effect is not restricted to only the nematode. With the ornamental swordtail fish, Xiphophorus helleri, and the South African clawed frog, Xenopus laevis, we present phenomenological evidence that slight feminization occurred when these vertebrate species were exposed to a synthetic humic substance, a condensation product of polyphenols. The slight feminization was dose dependent.
Suppression subtractive hybridisation (SSH) was used to generate cDNA libraries representing genes differentially-expressed in liver from male plaice (Pleuronectes platessa) exposed to ethynyl oestradiol (EE2). BLAST analysis and alignments of the clones with database sequence suggested at least three vitellogenin (VTG) genes and three zona radiata protein (ZRP) genes were represented. Clones with unique sequence (62 up-, 13 down-regulated) were arrayed as probes on nylon membranes to investigate temporal expression of oestrogen-responsive genes in experimental animals. Arrays were hybridised with radiolabelled cDNAs prepared from hepatic mRNA from animals treated with EE2 for various times upto 21 days and from treated animals transferred to clean water for upto a further 31 days. By day 21 of treatment 11 out of 17 probes from unidentified genes, 21/22 VTG, 13/14 ZRP, 2/2 liver aspartic proteinase (LAP) and 8/10 other gene sequences were induced by EE2 exposure. Of the down-regulated sequences, only three showed significant, decreased expression and these encode cytochrome b and two with cryptic functions. Based on the pattern of temporal response the up-regulated probes fell into two classes. Pattern A reached maximum expression by day 16 of exposure and then declined prior to removal of EE2 at 21 days. Pattern B genes reached maximal expression between day 16 and 22, declining only after removal of EE2. Independent investigation of the expression patterns of selected probes using quantitative Real-Time PCR reproduced the distinctive patterns. The results indicate a previously unrecognised mechanism for oestrogenic toxicity in which there is a selective down-regulation of some egg proteins, potentially diminishing the quality of eggs and this may contribute to reproductive failure described elsewhere.
Although over 80 cytochrome P450 (CYP) encoding genes have been identified in the genome of the nematode Caenorhabditis elegans very little is known about their involvement in biotransformation. This paper demonstrates a concentration-dependent relationship of C. elegans CYP35A1, A2, A5, and C1 gene expression in response to four organic xenobiotics, namely atrazine, PCB52, fluoranthene, and lansoprazole. The toxicity of these xenobiotics was determined using a reproduction assay. CYP-specific messenger RNA expression was analyzed by semi-quantitative RT-PCR resulting in a strongly increasing, concentration-dependent induction well below the EC50 for reproduction. For PCB52, approximately 0.5% of the EC50 induces a 2-fold increase of CYP35 gene expression. Using a double mutant and multiple RNAi of CYP35A/C it was possible to diminish the reproduction decline caused by PCB52 and fluoranthene.
We report the effects of the hepatotoxic compound carbon tetrachloride (CCl(4)) and pyrene, a model polycyclic aromatic hydrocarbon, on the transcriptomes of juvenile rainbow trout kidneys and livers. Fish were exposed to sublethal doses for 4 days and expression of 1273 genes was measured using a cDNA microarray. Efforts were focused on differentiating between unspecific responses and those that can be regarded as molecular signatures of CCl(4) and pyrene toxicities. Expression profiles were analyzed in terms of Gene Ontology categories. Universal reactions to chemical toxicity were observed in metallothionein, HSP90 and mitochondrial proteins of oxidative phosphorylation, which were induced in both tissues. Several genes showed similar responses to both compounds in either kidney or liver; most of the effects are implicated in hematopoiesis and immune response. Stimulation of mitochondrial and heat shock proteins was greater in the liver than in the kidney, whereas genes involved in transcription, humoral immune response and apoptosis were suppressed. Pyrene and CCl(4) caused opposite effects on expression of several genes, including HSP-27, macrophage receptor Marco, metalloproteinases (MMP9 and MMP13), and delta-6 fatty acid desaturase. Pyrene affected mainly genes implicated in the maintenance of the genetic apparatus, immune response, glycolysis, and iron homeostasis. CCl(4) affected the structural proteins and genes involved in cellular stress, protein folding, and steroid metabolism. Overall, pyrene suppressed a range of protective or acclimative reactions, many of which were stimulated with CCl(4). Additionally, gene profiling analyses indicated adaptive and potentially maladaptive reactions to toxicity. For instance, stimulation of mitochondrial proteins coincided with suppression of catalase, whereas CCl(4) down-regulated fatty acid metabolism and peroxisomal proteins. A number of candidate biomarkers for ecotoxicological risk assessment were identified as our understanding of mechanisms of pyrene and CCl(4) toxicities in rainbow trout increased.