Endocrine Disruption in the Omics Era: New Views, New Hazards, New Approaches

INTRODUCTION - THE "OMICS" PERSPECTIVE

The completion of the Human Genome Project in 2001 represented a complete turning point in Biology. Together with the complete genomes of the yeast Saccharomyces cerevisiae (1996), the nematode Caenorhabditis elegans (1998), the fruitfly Drosophila melanogaster (2000), the cress Arabidopsis thaliana, and the fish Takifugu rubripes (2002), it allowed for the first time the analysis of the genetic makeup of Eukaryotes in its complete extension and to establish new functional, evolutive and physiological correlations between taxa, individuals, organs and cell types. The development of new highly efficient analytical techniques has allowed similar holistic approaches for essentially all components of the live cell. For example, following the biological flow of information, high throughput techniques of specific RNA quantitation (microarrays) allowed the description of the mRNA complement of a given cell or tissue, i.e., the description of its transcriptome. A similar approach, but using completely different methods (2D electrophoresis, advanced mass spectrometry techniques), has been applied to elucidate the protein composition (the "proteome") with unprecedented precision, whereas several analytical methodologies (gas and liquid chromatography coupled to mass spectrometry, capillary electrophoresis, and high resolution nuclear magnetic resonance (NMR) allow the description of the chemical composition of the cell, that is, the nature and composition of its metabolites (the "metabolome"). Fig. (1) summarizes the nature and challenges of each one of these "omic" analyses.

The application of “omics” tools/technologies is now widely employed in many research areas, from medicine to environmental sciences, as they give information on some key regulators of various processes in living organisms. They also represent a tremendous opportunity to improve human and wildlife health by the characterization of the environmental elements that impact public and wildlife health [1]. The recognition of these challenges and opportunities, along with the fact that many of the most prevalent diseases are associated with the endocrine system, has led to a focus on chemical exposures and especially endocrine disruptors. The term ecotoxicogenomics refers to the integration of genomic-based science into ecotoxicology using DNA array-based technologies, proteome and metabolomic analyses [2]. The approach has been fundamental in several aspects of our current comprehension of the effects of endocrine disruption: The elucidation of potential effects on different animal taxa, thanks to comparative genomics, the comprehension of the multiple targets for exogenous ligands in the cell, and the development of new and more precise methodologies to monitor endocrine disruption both in humans and in wildlife.

HORMONES AND PHYSIOLOGICAL PROCESSES

Hormone are endogenous molecules secreted by endocrine glands that travel through the blood stream and induce physiological effects on distant cells and tissues [3]. The mechanism by which hormones exert their actions on target cells remained obscure for decades, although the concept of the existence of an endogenous receptor responsible for the recognition of the hormone (the "ligand") and the triggering of the physiological changes derived from its presence dates from the early 70's [4]. The isolation and cloning of the first hormone receptors in the 80's opened the Molecular Endocrinology era [5, 6]. However, the recognition of the amazing complexity and implication of the hormonal signaling did not appear until the advancement of whole genome sequencing techniques and its extension to evolutionary distant species [6, 7]. Analysis of complete genomes of different species revealed the presence of dozens, if not hundreds, of evolutionary related proteins for which no ligand or function was known. These so-called "orphan" receptors are widespread across Metazoa and their functional characterization implicated a major progress on the understanding of cell regulation, development and even evolutionary links between animal taxa [6-8]. An overview of the different receptor families, their physiological ligands and their distribution among the major Metazoan taxa is shown in Table 1.

ENDOCRINE DISRUPTION

Environmental pollutants in the various ecosystems are a major concern worldwide. Our understanding of the potential adverse effects of anthropogenic contaminants has driven to an increasing public awareness on their influence on health and well-being of both humans and wildlife [3, 9]. The primary observed effects of those pollutants are related to several developmental and reproductive disorders in wildlife species, which have been clearly linked to environmental compounds that act as endocrine disrupting chemicals (EDCs) [3].

By definition “An endocrine disruptor is an exogenous substance or mixture that alters function(s) of the endocrine system and consequently causes adverse health effects in an intact organism, or its progeny, or (sub) populations" [10]. Reports of adverse effects by EDCs in the last decades include reproductive impairment (decrease fertility, hermafroditism and sex reversal, altered sex ratios, hatching success), metabolic defects (Thyroid dysfunction, body weight and fat tissue control alteration), and alteration of immunological and behavioural functions have been observed in wild populations of mammals, birds, reptiles, amphibians, fish and mollusks; epidemiological evidences indicate that at least part of these or similar effects may be already occurring in human populations [3, 10-14].

There are varied sources of environmental contaminants that may disrupt the endocrine system (Fig. 2). The human exposure typically occurs with the environmental contamination of the food chain, especially fresh water fish and meat, contact with contaminated household dust, and occupational exposure [9]. Some chemicals were banned or removed from production years ago but persist in the environment. On the other hand other EDCs are high production volume chemicals found in a many household products. Bisphenol A (BPA), for example, is present in polycarbonate plastics, including beverage and food storage containers; epoxy resins that line the interior of metal cans, and in the ink used for thermal paper receipts. Many textiles contain contaminants, such as flame-retardants, including tetrabromobisphenol A and polybrominated diphenyl ethers. Some individuals have also been exposed to contaminants with adverse effects as a result of medical (diethylstilbestrol; DES), dental (diglycidyl methacrylate; GMA) or dietary (phytoestrogens) interventions. Urban wastewaters are important pollutant sources of natural and synthetic estrogens and other hormones that are subsequently found in surface waters [15, 16]. Thus, exposure to EDCs is ubiquitous and inevitable and there is growing concern that living in an EDC contaminated world may be contributing to adverse health trends, such as early puberty and infertility, because of growing evidence that a number of EDCs can produce varied effects [3, 17], (Fig. 2).

EVOLUTIONARY PERSPECTIVE OF ENDOCRINE DISRUPTION: EDCS IN INVERTEBRATES

From its very first origins, the concept of endocrine disruption has been deeply associated to terrestrial vertebrate wildlife (birds, reptiles) and humans. However, environmental and laboratory research indicate that fish species are particularly sensitive to many forms of endocrine disruption, particularly to estrogens [18]. Finally, the discovery that the presence of TBT in coastal waters is linked to imposex in marine gastropods brought the same concept to mollusks and invertebrates in general [19, 20]. The more recent deleterious effects of neonicotinoid insecticides on bee colonies around the world is probably a further example of the importance of recognizing and controlling environmental EDCs than can effect non-vertebrate species [21]. Nevertheless, information on EDCs in invertebrate species is still limited [22-30].

Difficulties in performing invertebrate studies are largely due to the limited knowledge on the endocrine physiology of many invertebrate groups that represent important components of ecosystems. In the first place, the concept "invertebrate" is essentially instrumental, as it covers species from widely different Metazoans. Only among animals with bilateral symmetry (Bilateria), three major taxonomic clades appear: Deuterostomata (which includes Vertebrates and Echinoderms, among others), Ecdisozoa (Nematodes and Arthropods) and Spiralia/Lophotrochozoa (Mollusks and Annelids), the two later forming the group of Protostomata [31, 32] (Fig. 3). These major groups of animals, as well as the ancestral groups of Porifera and Ctenophora, are characterized by their distinct embryonic developments. Last advances on biochemistry, genome sequences and regulatory studies showed that they exhibit fundamental differences in their complements of nuclear receptors, in their ability to synthesize and metabolize molecules with ligand activity, and, most probably, in the physiological roles of these molecules (Fig. 3, Table 1) [8, 31-34].

Despite the great invertebrate genetic and taxonomic diversity, ecotoxicological studies are limited to few worms, mollusks and arthropod species that are easy to maintain and rear in the lab. Furthermore, in regard of their endocrine system, invertebrates have been relatively far less studied than vertebrates, with most of the literature published on invertebrate endocrinology referring to mollusks, insects and crustaceans [19, 34, 35]. When dealing with transcriptomic or proteomic analyses the situation is even worse, because of the relatively low number of invertebrate genomes at least partially sequenced and more or less correctly annotated [36, 37]. For these reasons there is a high degree of uncertainty to relate an adverse effect on invertebrate growth or reproduction to specific changes of its endocrine system. For example, despite the abundance of literature dealing with the development, growth and reproductive effects of EDC in invertebrates, only few studies have assessed unambiguously a truly endocrine disrupting effect [38]. Several studies tested the wrong premise that mammalian or vertebrate EDC should act as endocrine disruptors in invertebrates through identical or at least homologous mechanisms of action. This was especially evident for estrogenic/androgenic compounds tested against different arthropod species, which lack functional estrogenic/androgenic receptors (Table 1). In many cases, the detrimental effects of EDC on growth and reproduction reported, were related to egg mortality or feeding inhibition rather than to effects on endocrine disruption.

Research conducted in the crustacean species Daphnia provides conclusive evidence that juvenile hormone agonists enhanced male production disrupting the ultraspiracle receptor signaling pathway during the initial phases of embryo development [39]. There is also evidence that juvenile hormone agonists modulate ecdysteroid activity causing embryo arrest or abnormalities throughout the ultraspiracle- and ecdysone-receptor complex [40]. Studies on the effects of antidepressants show that molluscan and crustacean reproductive and locomotion systems are affected by antidepressants at environmentally relevant concentrations [35].

In particular, antidepressants affect spawning and larval release in bivalves and disrupt locomotion and reduce fecundity in snails. In crustaceans, antidepressants affect freshwater amphipod activity patterns, marine amphipod photo and geotactic behavior, crayfish aggression, and daphnid reproduction and development [34, 35, 41]. The above reported effects are likely to be related through out disruption of neuroendocrine signaling pathways. Like in vertebrates, the endocrine control of growth, reproduction and behavior in invertebrates are initiated by neurohormones [34, 35, 41]. Serotonin and antidepressants targeting these neurohormones induce spawning in bivalves, alter locomotion and foraging behavior in gastropods and alter mimetic and predatory behavior and memory in cephalopods [42-49].

Neurohormones control a wide variety of biological systems in crustaceans, including reproduction, growth, maturation, larval development, immune function, metabolism, behavior and colour physiology. For example, both serotonin and dopamine have been found to stimulate the release of multiple other crustacean neuropeptide hormones including hyperglycaemic hormone, red and black pigment dispersing/concentrating hormone, neurodepressing hormone, molt-inhibiting hormone and gonad-stimulating hormone [34, 41, 50, 51] (Fig. 4). Although neurohormonal disruption in molluscan and crustacean species have been mostly limited to antidepressants, there are other pharmaceutical drugs targeting neuronal receptors or other enzymes that may also alter neuroendocrinological pathways that regulate key physiological function. One of those are non-steroidal anti-inflammatory drugs (NSAIDs) that interrupt crustacean eicosanoid metabolism, which appears to disrupt signal transduction affecting juvenile hormone metabolism and oogenesis [52].

The implementation of omics approaches to invertebrates is limited to only few model species and studies that have addressed omics and EDCs are scarce [36, 37, 53-56]. Probably the best characterized and solidly established mode of action (MoA) of "canonical" (i.e., active in vertebrates) EDCs is the disruption of the enzymatic pathways for steroid synthesis, which are very well conserved within Metazoans [19, 57, 58]. However, several studies [53, 54, 56, 59-63] reported disruption on regulatory mechanisms in addition to the effects on the steroidogenic pathway. Transcriptomic patterns of intersex specimens of clams Scrobicularia plana showed a deregulation of the androgen receptor signaling pathway [53], an effect also described for Mya arenaria males exposed to TBT [64]. Metabolomic and proteomic analyses revealed different cases of neuroendocrine disruption in crustaceans and mollusks. For example, atrazine and its metabolites affected eicosanoids in the isopode Hyalella azteca suggesting possible perturbations in neuropeptide hormonal systems [55]. Similarly, ibuprofen inhibits reproduction in the crustacean Daphnia magna due to the de-regulation of the ecosanoid signaling pathway, and hence of prostaglandins, which in crustaceans control reproduction [62, 65]. A typical vertebrate estrogenic EDC, ethinylestradiol, alters metabolite pathways related to energy reserves, signal transduction, immune response, and neuromodulation in the unionid mussel Lampsilis fasciola. These effects result in physiological changes, as altered siphon and mantle [54], both in male and females. Conversely, the poly-bromo-diphenyl ether congener (BDE 47) affects differentially male and female specimens of the marine mussel Mytilus galloprovincialis: males show effects in energy metabolism, whereas, in females, BDE 47 disrupts both osmotic regulation and energy metabolism [56].

Very likely, we are just beginning to understand the phenomenon of EDC in invertebrates, and many of the chemicals that are contaminating terrestrial, freshwater and marine ecosystem may have endocrine disrupting effects on invertebrate species. Given the vast genetic diversity of Metazoans, the MoA already defined for vertebrates may or may not apply to any particular taxon. Therefore, EDC toxic effects may have unanticipated, and many times unnoticed until they reach global scale, deleterious effects in the ecosystems that depend upon invertebrate populations.

OMICS’ APPLICATION ON EDCS ASSESSMENT

A Practical Approach: Predictive Endocrine Disruption

Most international regulators posed as a major objective to stop the continuous deterioration of the environment by the thousands of new products entering into the marked each year. This objective demands multi-level, multi-tiered approaches, combining chemical analytical and biological methods [73]. Whereas the chemical identification of pollutants has advanced notoriously in recent years, allowing quantification of a wide array of potential pollutants in the environment at sub-ppb levels, the biological characterization of their putative effects on ecosystems and human health is clearly lagging behind in terms of defining new risks ("emerging pollutants"), identifying emerging effects of classical pollutants (behaviour, neurological, immunological or metabolic disorders, for example) or characterizing the mechanism of toxicity [74]. This is a most serious impediment for both the correct evaluation of environmental impacts and for the assessment of risks associated to new substances.

Toxicologists (including Ecotoxicologists) have developed different methods to evaluate the toxicity of new and well-known substances in order to evaluate their possible environmental and human health hazards either before they enter the marked or before their impact in human populations and wildlife exceeds their proved or assumed economic benefits. Classic toxicology relies heavily on the use of standard animal models to evaluate toxic effects, including rodents and dogs as surrogate for human populations, and fish, crustaceans, and algae to evaluate environmental hazards. This approach has two major limitations. First, they are slow and costly, taking several months and thousands of dollars to evaluate a single substance. And second, there is a quasi-universal claim to reduce animal suffering to the absolutely unavoidable minimum, by applying what is known as the 3Rs rules: replacement, reduction and refinement of animal experiments, [75]. Cell cultures, non-animal or non-vertebrate models (yeasts, crustaceans, annelida, nematodes), and free embryos before their autonomous stage have been proposed as valid alternatives to conventional animal testing for both toxicology and food and drug safety studies [76].

Within the general context of toxicology, endocrine disruption presents a further difficulty in their characterization in non-vertebrate models. For one thing, different animal taxa may encompass different complements of receptors, and therefore their susceptibility to a particular substance may differ wildly. In addition, the analysis of binding to a particular receptor, even accounting for species specificity, may not suffice to characterize an endocrine disruptor as such. Many estrogenic compounds do interact with the ER and, therefore, they are recognizable by single-cell or even in vitro or QSAR approaches. But this does not apply to those that affect steroid metabolism or others, like HCB, for which a suitable model of action has not been demonstrated [77]. And these problems increase when more "metabolic" signaling pathways, like thyroid, PPAR, retinoids, and others, in which the so-called non-genomic effects (effects on hormone metabolism, transport or degradation, neuroendocrine control, etc.) may easily shadow the more "classic", receptor-binding linked effects [57, 77-80].

Genomic and other omic techniques can be used to infer complex toxic effects, affecting higher levels of organization, from the analysis of a reduced number of well-controlled model species. The basic idea underlying this approach is that the signaling pathways are well conserved during evolution, so the determination of the effect of a given substance on a specific pathway on a model species is transferrable to humans or environmentally relevant species as long as the modes of action are known and the pathways from the molecular initiating effects and the final adverse effects are conserved. This translation of toxic effects through the different levels of biological organization is known as Adverse outcome pathway (AOP) analysis. In this approach, the different organization levels are analyzed, ranking the corresponding data according to their ecological relevance and its predictive capacity and mechanistic utility (Fig. 5) [69, 81]. Once identified, AOPs can be used to develop high throughput omic technology-based predictive assays, providing valuable information for human and environmental risk assessment. By comparing AOPs across species is possible to identify specific and shared mechanisms of toxicity, and hence use this information in understanding and managing environmental risks. Fig. (6) shows an example of the AOP approach and of its application in two cases of endocrine disruption, one in fish and a second in Daphnia [82, 83]. Note the central part of omic technologies in the process of ellucidating the mechanisms of toxicity for each type of disruption (ectopic expression of female proteins in males, disruption of carbohydrate metabolism).

Concluding Remarks: The Need for a Synthetic Approach

The "genome revolution" has modified forever the fundamentals of Biology. For the first time, quantitative approaches can be applied and modeled, using truly holistic points of view. In the field of Ecotoxicology, and particularly in the study of endocrine disruption, the knowledge of complete genomes from very different taxa discovered not only scores of new potential targets in the already know organisms, but also to predict or explain unsuspected effects in non-targeted, less known species of ecological significance.

The ever-increasing importance of the omic technologies in Ecotoxicology resulted in an overflow of data from multiple techniques and approaches that need to be analyzed, integrated and presented in a useful way to understand the implications at individual, population and ecosystem levels. AOP analyses are useful to tell apart, as well to correlate one which other, the initial (or "key") events of the toxic effects (i.e., endocrine disruption) and the truly adverse effects, those that compromise the health of the individuals, populations, and finally the ecosystems.

SUPPLEMENTARY MATERIAL

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