Expert Views :: Signals, Pseudo-Signals, Parasites And Noise: The Many Ways To Disruption Of Biological Communications Paul Galand At the 2000 e.hormone meeting, science philosopher Sheldon Krimsky raised the question of the lack of a general theory about endocrine disruption. During the passionate and stimulating debate that followed, I took the opposite view that knowledge and concepts from several well-established fields in fact converge to support the concept, each with its appropriate theoretical framework. It is Woody Allen, I believe, who once said, "Making predictions is a very difficult task, especially regarding the future.” Yet, from what we know, it now appears rather surprising that no one seems to have foreseen that endocrine disruption would be ineluctable. There were, as John McLachlan pointed out in a recent review (Endocr. Reviews, 22, 319, 2001), predictions made by Roy Hertz regarding expectable estrogenic disruption due to the release in the environment of estrogens — including synthetic ones — used in cattle feed. However, what we knew for decades about the hormone-receptor systems and about biology in general, allowed us to expect different kinds of disruption; not only of estrogenic functions, but also of other hormone-receptor systems and their downstream network of signal transduction. Whereas this knowledge may be considered basic and evident, it seems that its theoretical implications, despite being no less evident, did not receive due attention. As I shall try to show, one could have predicted the perturbations provoked, not only by substances made to mimic or antagonize the endocrine system considered, but also by compounds — natural or anthropogenic — aimed at acting on quite different biological functions and even some without any biologically orientated aim (as for what McLachlan designated as "inadvertent” estrogens). About Semantics But before going further into this commentary, a few words are needed about semantics. It is a very common attitude, nowadays, in scientific milieus to refuse any discussion about definition of terms as “purely semantic.” This is the source of much confusion, misunderstanding and sterile debates. A most frequent one is the premature coining of “concept-charged” terms to designate newly discovered molecules or genes, based on the first function to which it was found or seemed associated. It then may sometimes become difficult to conceive that these substances might have quite other function(s), besides — or even rather than — those implicit to their initial designation. “Oncogene,” “EGF” or “heat shock proteins” are just examples among many. This said, if there is a field in which everybody would admit that the “semantic” perspective should not be neglected, this surely is the “environmental disrupters” of hormone signaling. After all, the “sign,” “the signal” (“sèmeion” in Greek), is exactly what “semantic” refers to. There is a wide diversity of possible ways to perturb endocrine function, possibly occurring at all of levels in the cascade of events that precede and follow hormonal signaling. Before considering the different types of mechanisms this may cover, a distinction must be made between perception — or sensing — of environmental properties by living organisms; and true signaling — or communication — between living entities. 1. Perception (or sensing) by living organisms of environmental characteristics, namely physical parameters (like cold, heat, mechanical properties) or chemical properties, is made through more or less elaborate and specific sensors. The latter may or may not interpret their perception as stimulus for providing more or less adaptive biological response(s), selected by evolution. These functions include exploring, identifying food and detecting chemical or physical adverse situations. (When this involves molecules, the sensor may have already properties that define them as receptors, a term I prefer to reserve for true biological communication, yet, keeping in mind that receptors probably evolved from sensors). 2. True Communication (or signaling) between living organisms or entities (individuals of different or same species or their constituent organs, tissues, cells or cellular compartments) is performed through "signals" or mimetic thereof (i.e. pseudo-signals). These are sent by the emitter (or producer) and recognized by specific receptors by the receiver. Their reception by sensory organs or, for molecules, their binding to specific receptors, triggers a more or less complex cascade of events that constitute the response. The "aim" of signals (i.e. the capacity they were selected for) is to influence the physiology and/or behavior of the receiver in a way somehow advantageous to the former or to both partners, individual or species reproductive fitness being the ultimate criterion for selection. Part or all of the response of the receiver may be opposed to the emitter's aim, which leads to what has been designated as the "arm race" between preys or hosts and their predators or parasites (see below). Disruption Thus, in these definitions a stimulus is not a signal as the environment never does "signal" anything, even when its influence is through compounds acting on one or more organisms, and despite this may be acting on one of the signal transduction devices of the latter. The sun does not signal anything when it rises, even though this may induce a cock to sing. This is not a purely academic point, as it introduces the need to distinguish between two types of negative interference in normal endocrine functions that combine to increase the frequency of the accidental occurrence of such interference. A first type consists in the “intentional” or accidental production of a true signal or of a mimics thereof — what I suggested to designate as a pseudo-signal — either natural or synthetic, i.e. a substance which binds to a receptor and either activates or inactivates it. Such a situation is somehow analogous to what occurs in radio when somebody intentionally or accidentally breaks a communication code and sends a true — yet untimely — or a false message. This, I suggest, should be designated "direct — or specific — endocrine disruption" in this included the agonistic or antagonistic effects that may result. Another type of perturbation of endocrine functions corresponds to the simple parasitic or noise effects of the background on a specific signal, either at its emission or at its reception and recognition (like, in radio, respectively, with interference by atmospherics or by a signal produced on a close wavelength and with perturbation of the reception by a noisy surrounding). This type of perturbations of endocrine functions, generally scored with those of the first type, might better be termed "indirect — or unspecific — endocrine disruption.” I shall not expatiate on the evident possibility that the default in endocrine functions may simply result from an overall negative effect on general physiology (the equivalent in radio, of destroying an element of the amplification stage or of simply cutting the source of energy). Neglecting this completely unspecific type of perturbation would lead to consider many things — the guillotine among others — as endocrine disrupter, based on the behavior of the subject after exposure to them. More directly relating to true endocrine perturbation, a given compound might block a hormonally controlled activity by acting well upstream or downstream of the reactions with receptors. LeBlanc, et al., perfectly illustrated this for testosterone by summing up available data about the effects of a variety of pesticides, some inhibiting testosterone synthesis, others favoring its degradation, thus adding their anti-masculinizing actions to the estrogenic or anti-androgenic properties of the same or of other pesticides. At the e.hormone 2001 conference, another example was proposed by Wenchao Song, namely the inhibition by polychlorinated biphenyl of the enzyme estrogen sulfotransferase, involved in the inactivation of estrogens. Similarly, TCD-dioxine seems able to augment the rate of elimination of the thyroid hormone T4 by stimulating hepatic UDP-glucosyltransferase. Conversely, blockade might as well occur downstream of the receptor and the related signaling cascade, as shown, for example, by the negative modulation of the GR transcriptional activity by arsenic. This may even occur farther downstream to signal transduction pathway, i.e. directly on the end point submitted to its control. A good illustration for this is the case of DDT negative effects on reproduction rate in birds of prey, so classical that it has become a paradigm in the field. Early studies established a link between presence in the environment of DDT derivatives and thinning of prey birds' eggshell, a still controversial conclusion which was attributed to the estrogenic potency of the pollutant. However, later studies indicated that this might be due, at least in part, to direct inhibition of the activity of the enzyme involved in calcium deposition. All this makes it evident that the capacity of a given substance to act as a "disrupter,” based on a given endpoint, would not necessarily relate to its ability to bind a receptor. Thus, evaluating the latter would not always constitute a valid biomarker of such disruption. It also explains, at least partly, an otherwise surprising fact, namely the high incidence of agents actually found to "disrupt" endocrine functions, despite the limited number of compounds that do exist; but the more so considering the very low number of them which have been accidentally or purportedly tested for such potential. This conclusion was indeed based on "pooled" data relevant to the different types of disruption I mentioned. As I'll try to show, other reasons, however, may contribute to explain the high incidence of direct, truly specific, endocrine disruption, involving an action at the level of the receptor itself. Estrogen Disruption: the tip of an iceberg? The possibility must be considered that available observations about sexual hormones systems might just represent the tip of the iceberg. Indeed, by considering the pleiotropic effects of estrogens on their target tissues as well as the multiplicity of target tissues, whether or not involved in primary and secondary sexual characters or behaviors, we disposed of a large target, i.e., a wide panel of criteria that greatly facilitated the detection of any abnormality at this level. On the other hand; if such events were detected first, this might be simply due to the fact that practically all somatic functions were selected on the basis of the reproductive advantage they procured. So that, practically, most of the somatic part of an animal might somehow be seen as an assembly of secondary sexual characters, contributing in one or another way to reproductive fitness. Whatever the level of a perturbation, it will finally affect reproductive physiology and/or behavior. This applies to simple non-specific damages provoked by a physical or a chemical factor as well as to the effects exerted on the controlling components of hormone signaling. This leaves open the possibility that disruption by environmental compounds of other hormone-receptor systems than the E-ER system might be equally frequent, but remained up to now undetected. Endocrine Disrupters and their Highly Predictable Occurrence The reasons for the high probability of existing and yet to be discovered cases of direct endocrine disruption are of different orders, including: • the radiating evolution of receptors according to what Jacob termed "evolution by tinkering" • the relatively small elements — sequence, structural conformation — on which is based their specific recognition of a natural cognate ligand and the apparent limit to the number of molecules which can be recognized as different by living organisms • the way specificity of cell or tissue responses is achieved • the existence of ligand–independent modes of receptor activation. Evolution of Receptors When new structures and functions appear during evolution, this often — if not always — consists in using former ones and remodeling, modifying them rather than creating new ones ab nihilo. This is what Jacob described as "nature's tinkering.” Classical examples are the formation of birds' wings from reptiles’ forelegs, or of the internal ear's bones from remodeling of mandible components. At the molecular level, and directly related to this discussion, we have the example of radiating evolution from a common molecular ancestor of entire families of receptors. The ancestor molecule specifically responded to physical agents or chemical compounds present in the external milieu, but was not necessarily a receptor, in the sense given to this term in endocrinology, even if it specifically recognized a substrate or a type of substrate. Since the first problems living organisms had to solve implied to perceive properties of their external milieu, some — if not all — receptors probably derived from molecules which, at the origin, acted as "sensors" (see above). An example is the VR1-vanilloid receptor type1 which responds to heat and — but only in mammals — also to anandamide, a lesion associated protein responsible for the heat sensation provoked by a wound. Another example is CMR1/TRPM8, a cold sensor, also acting as a receptor for menthol and responsible of the cool sensation felt when absorbing compounds of this class. Another heat sensor has been found, namely VR-1, which presents an analogy with VR1, but does not bind anandamide. That it might also be a receptor of some type of molecules was suggested by its discoverers, based on its presence in organs like lung, spleen or intestine, unlikely to be ever submitted to high temperatures in normal life. One interesting aspect about VR1 resides in that, in mammals, it also responds to capsaicin, the substance present in red pepper (Capsicum) and responsible for the heat effect associated with consumption of the latter. This is made possible by the presence in capsaicin of the same 8 amino acids sequence through which anandamide is recognized by the mammalian VR1. The plant thus uses to its advantage a property presented by the mammalian VR1, not by the avian one, thus making itself unpalatable to the former (who would completely digest the seeds) not to the birds, who fulfill an honest contract by dispersing seeds left intact. Two reflections on this: • First, that it is unlikely that the ancestors of red chili pepper purportedly produced a mimic of anandamide, but well that this was produced by accident — as a secondary metabolite, like for most of the chemical defenses — and that evolution exploited this by increasing its production and, probably, also by augmenting its affinity for the mutated mammalian VR1. • Second, the relatively short sequence on which "specific" recognition of anandamide was based, favored the accidental occurrence of and subsequent exploitation of a mimic. This suggests that the same kind of accident would also as easily occur with synthetic compound or sub-products, especially considering that what matters is not the sequence itself but the molecular conformation it confers to a given site. This might be put in relation with the nightmare of those who use immunological techniques, namely the existence of cross-reactions, by which compounds different — and sometimes much different — from the antigen against which it was raised react with an antibody, even monoclonal. Again, in this case, the size of the element determining specificity (here the antigenic determinant) is relatively small — a few aminoacids in a protein - and again, cross-recognition can occur with molecules otherwise completely unrelated chemically, some of them unlikely to have ever been met before by any animal, not to speak of the antibody-producing species. Together, this underlines that there is a limit to the number of molecular conformations that can absolutely be resolved by a biological device. In other words, that there is a limit to the degree of specificity. It ensues from this that compounds chemically unrelated to bioactive substances produced by biological systems or organisms may present enough conformational resemblance with them to mimic or interfere in their action by interacting with the receptor or with the particular type of molecule that they specifically recognize. This may also contribute to explain the otherwise surprising fact I mentioned above, namely the high incidence of agents found to "disrupt" endocrine functions, considering the relatively low number of them which have been accidentally or purportedly tested for such potential. About Specificity Consider, for example, the nuclear receptors. They form a large super-family of phylogenetically related proteins, endowed with a high affinity for hydrophobic molecules as diverse as steroid hormones, retinoic acids, thyroid hormones, leukotriènes or prostaglandines. On one hand, they present a highly conserved structure, this of course essentially reflecting part the similarity of their mode of action once they are activated. This nevertheless offers the possibility that at least some of the compounds affecting one member of the family might as well affect other members. On the other hand this common origin raises the interesting question of how specificity is achieved when a new receptor is formed. Theoretically, this of course implies that it recognizes its cognate ligand with high affinity, but not necessarily that it should have no — or low –affinity for any other type of ligand recognized by members of its family. It suffices that this lack of affinity applies to the ligands that the target cell considered is likely to meet in its normal life, these being dealt with by appropriate coexisting receptors. Along this line, it was otherwise indifferent to their function that they did or did not keep sequences that, in the ancestors of the species, bound to ligands that the target cell considered would normally never encounter any more. This may be because the species or left the milieu where these were found or because it developed a barrier or a clearance capacity against them. It may also be due to the fact that during differentiation, the receptor segregates in target cells out of reach of such compounds. If so, any situation which would re-establish contact with substances similar to those that were acting in the past on the parent molecule, might again be recognized by the more modern receptor, somehow "taken by surprise". Similarly, compounds might be synthesized that are endowed with properties similar to those of the unwanted ligand, but able to overcome the above-mentioned barriers. This is particularly likely for the receptor sequences that played a significant role in the sensor precursor, which may perhaps explain some types of "ligand-independent" receptor activation. Whatsoever, the lack of absolute specificity and the homologies between receptors — together with the above mentioned limit to the discriminatory power of biological molecules — made it highly predictable that compounds identical or similar to those responsible for observed disruption of ER functions would somehow interfere in the functioning of parent receptors like androgen, thyroid hormone or glucocorticoid receptors, as indeed observed. It must however be kept in mind that molecules playing a specific role in given organisms would not necessarily play the same one in distantly related organisms as with evolution, the molecular and cytological environment (in this comprised the overall network of genetic expression and structural elements) may have changed. The Red Queen Hypothesis and the arm race between species Since the origins, predators and parasites are engaged in a kind of war, in which arms (poisons, repellents, unpalatable compounds) and countermeasures are developed or which, to the contrary, leads to a kind of agreement, a deal beneficial to the two parties (as between nectar-feeding animals and the plants they pollinate). Besides the directly bioactive molecules involved in these interactions, this often is associated with the use of true signaling, i.e. signals produced by an emitter "to" influence in its favor the behavior or functions of a receiver. These might consist in advertising toxicity or dangerousness (aposematic signals) or in luring potential preys by mimicking the signals they use for intra-specific communication, based on physical cues (form, colors, behavior, bioluminescent flashes) but also on chemical compounds (attractants, pheromones mimics, etc.). Those signals may use the sensory system and, by this, either subvert behavioral programs normally responding to stimuli meaningful to the recipient species or, when consisting in chemical components, be recognized by specific receptors of which they imitate the normal ligand. The Red Queen hypothesis assumes that both camps evolve under the selection pressure thus alternatively created. In such a way that — like Alice's Red Queen, who had to run all the time to stay in the same place in her moving world — both camps are engaged in a continuous genetic race that maintains their relationship. There is a long story on Earth of organisms producing compounds which directly interfere with other ones' functions or communication systems, i.e. in one way or another, with their receptors and/or cascades of signal transduction, as well as with specific steps in their metabolic pathways. In parallel, organisms responded to this in various ways so as to preserve their reproductive capacity. This mutual adaptation — co-evolution — of preys or hosts and predators or parasites, was possible because it was progressive on both sides, thus compatible with the time of response of the genomes involved, in terms of mutation rate. One should not expect that this would necessarily be the same for what concerns the high rate of dumping of an increasing variety of synthetic compounds, some intentionally produced for their bioactivity, some accidentally endowed with such properties. On the other hand, nature's experience in producing biologically active compounds indicated that compounds acting in — or on — a living organism might also act, but not necessarily in the same way, on other living beings, even evolutionary distant from the normal target species. This was at the basis of the "molecular ecology" approach in pharmacological research and indeed brought positive results, to the extent that Caporale estimated in 1995 that "All of the drugs discovered at the Merck Research Laboratories that became available to patients in the last decade emerged from programs that benefited from knowledge of biological diversity.” It would therefore seemed paradoxical that the same companies which engage this approach would consider endocrine disruption as unlikely to occur, especially with compounds — biocides or pesticides — produced with the aim to affect biological functions of some living organisms. Ligand–independent Activation of a Receptor Among the elements that increase this likelihood of endocrine disruption, a place should be given to ligand-independent activation of the receptor. This has abundantly been documented for the ER, which can be induced to acquire an activated conformation under the action of a diversity of factors, including IGF, tRA or EGF. It was shown in the latter case that this entails direct phosphorylation of the appropriate sites on the ER. This opens a large array of possible direct or indirect disruption of the E-ER system as well as of other parent endocrine systems, via an action on such direct, ligand-independent mode of activation of the receptor receptors of the ER. For example, this might account for the observation that some PCBs may activate the thyroid receptor, without seemingly binding to it. If this occurs, the functions thus perturbed would of course include those by which the considered hormone normally modulates the E-ER system, which adds a further potential mechanism of disruption of the latter involving no direct action of the disrupter on the ER. Conclusion Rather than being in need for a theoretical framework in which to inscribe endocrine disruption, several fields, each with its own conceptual basis, would support this concept, if available facts did not already allow the conclusion that it is more than a hypothetical view. Somehow paradoxically, it results from above considerations that looking for disruptions of estrogen or androgen functions might as well reveal that they are due to disruption of other endocrine systems. When looking for biomarkers of possible presence of endocrine disrupters, one should keep in mind the diversity of levels, more or less specific to the endocrine signaling pathways, at which disruption is susceptible to occur. Keep in mind, among other things, that this does not necessarily imply affinity for the concerned receptor (no more, as underlined by McLachlan in his above mentioned review, that such binding necessarily implies disruption). Based on the above considerations I am convinced that a close look at any endocrine system — or by the way at any ligand-specific system — will unavoidably reveal, in a variety of species, the action of environmental disrupters, some — but not all — of which, already known to perturb the estrogenic functions. This statement under the form of a prediction will be my conclusion.