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Computer Based Strengths and Weaknesses

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Of all of TiPED’s five testing tiers, the computer-based modeling is the least developed.  In an ideal world, one would be able to enter a molecular structure into a computer and have an instant read on its potential endocrine activity.  Unfortunately, such tools are not yet readily available and what tools exist are not accurate enough to stand alone.  That said, progress is being made toward this ultimate goal. For instance:

Making predictions about how a molecule will act based on quantitative structure activity relationships or QSAR:

Although it is potentially a useful statistical tool, obtaining a meaningful Q/SAR predictive model on toxicity is problematic, and depends on several factors, including the quality and availability of biological data, the statistical methods employed, and the choice of descriptors. A useful Q/SAR model would incorporate the following characteristics:

1) Include a training set comprised of a sufficient number of molecules that cover the range of properties to be predicted by the model.

2) The number of compounds in the training set should be far more numerous (at least 5 to 10 fold) than the number of non-correlated descriptors used to calculate the model. Furthermore, the descriptors should be biophysically relevant to the property being predicted.

3) The model should be applicable to novel compounds and allow for mechanistic information related to the endpoint of interest.

4) Preferably, the simplest model should be selected.

For the purposes here, a chemist should consider the following limitations of the Q/SAR approach when selecting a Tier 1 method to predict EDC potential:

  • The “SAR Paradox”, the fact that molecules of similar structure often have very dissimilar biological activity[16].
  • Each Q/SAR model predicts a specific endpoint, and only for chemicals with the identical mechanism.
  • Q/SAR models do not perform well with chemical structures outside the training set.
  • Most nuclear receptors have not been the focus of Q/SAR modeling, and there almost certainly are receptors yet to discover. Existing Q/SAR models predict only a subset of potential endocrine-activity and as such are insufficient.
  • Q/SAR models do not predict whether the compound agonizes or antagonizes a receptor.
  • Care must be taken to avoid deriving an over-fitted model (e.g. one that describes random error or noise, rather than an underlying relationship) and generating useless interpretations of structural/molecular data.

In sum, while Q/SAR models currently can be used as statistical tools for broad statements of probability they are not yet sufficiently developed for predictive toxicology, especially for endocrine disruption; additional tools must be used to provide a fuller picture.

Modeling of Biological Activity (Pocket Modeling, Molecular Docking):

The simplest way to think about a molecule and its receptor is to picture them as a lock and key, with a caveat that both of them are somewhat flexible.  In a molecular docking model, the goal is to determine the correct orientation and adjustments of these two components. Specifically, molecular docking predicts the preferred orientation a molecule will adopt when bound to another molecule (i.e. the receptor) to form a stable complex. This information can be used to predict the binding affinity, or strength of association between the two molecules. Because the relative orientation of two molecules influences whether agonism or antagonism of the receptor results from their interaction, this method is useful for determining what type of signal a novel chemical is predicted to generate at the receptor. The limitation of this approach is that the molecular docking method requires an available crystal structure of the ligand-binding domain of interest, or at least of its close relative, as well as understanding of the domain’s flexibility, and structures being altered by residence in different cellular locations, such as plasma membrane vs. aqueous compartments.

The main approach used by scientists that study molecular docking simulates the actual docking process, whereby the ligand moves into position within the receptor’s active site following a series of rigid body transformations and internal changes to the ligand structure, such as torsion angle rotations, as well as changes in the binding pocket structure [12]. Unlike simple comparisons of the complementarity of receptor and ligand shapes, simulation approaches can incorporate both ligand and receptor flexibility into the model, thus it is more reflective of what actually happens during ligand-receptor interactions. A disadvantage of this approach is that it is more time-consuming.

Molecular docking modeling tools have been developed in connection with pharmaceutical chemistry and are now being adapted to predict endocrine disruption potential.  Initial studies have demonstrated the acute accuracy of the tool, e.g. accurately modeling the interaction of polybrominated diphenyl ethers (PBDEs) with the ER[17, 18] and AR[19], as well as preliminary studies of a panel of NRs with crystallographic structures[20].  Recent tests of PPARγ models demonstrate the very strong (at close to 100% accuracy) discriminating ability of the docking models.  As this particular tool is further developed and refined, its utility in predicting EDCs will become extremely valuable as part of this tier in the TiPED toolbox.

Fish and Amphibian Assays

Assay Fish species Endpoints Reference(s)
Corticosteroid secretion Oncorhynchus mykiss Corticosteroid secretion in response to ACTH [106]
Rapid developmental toxicity HTS (in Tier 2) Zebrafish Morphological endpoints (edema, bent body axes, pigmentation anomalies, and organ malformations) [40, 107-111]
Fish sex development test Fathead minnow

 

Medaka

 

Zebrafish

 Designed to detect (anti-) estrogenic and (anti-) androgenic effects. Animals are exposed to test chemical before the onset of sexual differentiation.

Vitellogenin induction in males/inhibition in females.

Gonadal histopathology

Hormone levels

Sex ratio

Development of intersex

 [112]

 

[113]

 

[114]
Fish Two Generation Assay Fathead minnow

Medaka

Zebrafish

 Whole body, serum, tissue T4 levels [115]
Locomotion medium throughput assay (in Tier 2) Zebrafish Can identify subtle developmental abnormalities between the nervous and musculoskeletal systems [116]
Sex specific behavior Zebrafish Sex specific behaviors (aggressive: nipping, chasing, circling, avoiding, and reproductive: female association, spawning, chasing, and nipping) [117]
Short-term reproduction assay/ 21-day fish assay Fathead minnow

Medaka

 Designed to detect (anti-) estrogenic and (anti-) androgenic effects. Mature male and female fish will be monitored during a 21-day chemical exposure; survival, reproductive behavior, and secondary sexual characteristics will be observed while fecundity and fertilization success will be monitored daily. At termination of the assay, measurements will be made of a number of endpoints reflective of the status of the reproductive endocrine system, including the GSI, gonadal histology, and plasma concentrations of vitellogenin. [115], [118];

[119], [120]
Transgenic reporter lines Zebrafish Current lines can detect estrogenic activity and aromatase induction. More transgenic lines are being developed.  

[41, 121, 122]
Assay Amphibian species Endpoints Reference(s)
Corticosteroid secretion X. laevis

Rana catesbeiana

 Corticosteroid secretion in response to ACTH [123]
SEXDAMAX X. laevis

X. tropicalis

 Sexual differentiation

Metamorphosis

 [124]

[125]

 

Targeted Cell Strengths and Weakness

Strengths: Endocrine disruptors are chemicals that interfere with the hormone system. Disruption in regulatory hormonal systems can cause devastating long-term effects such as problems in brain, metabolism, and disease states such as diabetes and autoimmune dysfunction. It may be of interest to test a group of new chemicals for potential endocrine disrupting activity before they are released for public use. Assays in this tier allow for the opportunity to further explore findings from computer based assessment by means of targeted nuclear receptor assays.

Target Cell assays allow: a) direct testing for possible disruption of biological signaling pathways by new compounds, b) to refine the findings from computer based toxicity predictions and pinpoint the endocrine systems that may be affected by the new compounds, and c) more efficiently target in vivo testing in the Cell Processes tier .

Additionally, one can also test a large number of chemicals at once using rapid, robotic tools.  These “high-throughput screens” (HTS) give relatively fast information and, because they can simultaneously test such a  large number of chemicals, can be relatively cost-efficient. HTS are now available using cell-based and cell-free methods. The two primary examples in development in the U.S. are ToxCast at the U.S. E.P.A.21; and Tox21, a joint effort U.S. E.P.A., National Institutes of Environmental Health Sciences/National Toxicology Program, National Institutes of Health and the Food and Drug Administration22.

Weaknesses:

The current design of the Targeted Cell battery of assays will allow one to obtain information about the ability of chemicals to interact with any one of 48 different nuclear receptors. However, some chemicals can interfere with hormone action in the body by mechanisms that do not target these receptors.  For example, some chemicals can activate enzyme systems in the body that can block hormone synthesis or metabolism.  In addition, chemicals may be modified in the body to form compounds that directly interact with receptors despite no such activity in the parent compounds.  Evaluating metabolism is important, but is not present in current assays in this tier tests.  Future iterations of targeted cell tier tests will include metabolism as a component, but this is not a trivial issue and will require development.

Additionally, the technologies employed in this tier make use of recombinant nuclear receptors that, in some cases, are fragments of the receptor. These technologies have the advantage of being relative inexpensive and rapid, but they can produce a higher level of false positive and false negative results.  This weakness is compensated for to some extent by having some redundancy in the battery of assays.  However, false positives would also be identified in Cell Processes assays as well as higher level assays.

As an overall concern, the high-throughput screens (HTS) in common use were originally developed for use in drug discovery. They work well at detecting pharmacologically-active compounds with strong effects. However, HTS were not initially designed (or performed by most labs) to detect weak activities, and it is precisely these weaker signals that may be biologically relevant and indicative of EDC activity. Hence care must be exercised in HTS use and interpretation for detecting EDC activity.

Mammalian Assay – Strengths and Weaknesses

This tier involves testing chemical candidates on a range of physiological and behavioral effects in a rat or mouse model.  This tier has several unique qualities that make it critical for assessing chemicals for potential EDC effects. While a chemist may wish to use any of other tiers as an entry point for initial screens, the Mammalian tier should usually be reserved only for the final level of testing due to the greater complexity of the experimental design, the need for a developmental model that spans several months, and our respect for minimizing the use of mammals for testing unless absolutely necessary.

Advantages:

If a chemical passes screening in earlier Tiers, it is important to determine its effects in a mammalian model. Rodents are recommended due to their high conservation of endocrine and neurobiological systems with other species, including humans. In addition, both rat and mouse models for EDCs are well studied and validated in the literature. They undergo relatively rapid postnatal development, they reach maturity in approximately 2 months or less, and they have short gestations (18-22 days). Other tiers use cells and cells lines, as well as non-mammalian testing protocols, which are highly relevant to human health. However, no other tier has the unique features of internal pregnancy in a uterine environment, followed by lactation and maternal care. Considering that most researchers believe the greatest cause for concern for EDC exposures is to developing fetuses and in infancy, which are critical developmental periods, such a mammalian model is needed.

The principal goal of the Mammalian tier is to assess the long-term health effects of early life exposure to the chemical of interest.  Tests are designed to maximize the likelihood of identifying any effects on overall growth and development, pubertal timing, behavior, sexual dimorphisms, immune function, and other life history endpoints that are not possible to comprehensively assess in silico, in vitro, or in non-mammalian animal models.

An advantage of the Mammalian Tier is its malleability and flexibility, depending on the information needed.  For example, if a compound displayed no EDC activity in the other tiers, this tier could be used to obtain confirmatory data from a mammalian model.  Alternatively, this tier could be used to more comprehensively assess the endocrine disrupting potential of a compound for which results in other tiers were difficult to interpret.  The endpoints chosen will thus necessarily differ depending on the type of information needed and the performance of the compound in the other tiers.

For some compounds, a guideline study using a rat model may be required as part of routine risk assessment and toxicity testing.  Unfortunately most of the government guideline studies as currently proposed contain an insufficient number of endocrine sensitive endpoints and do not include doses that are considered to be “environmentally relevant” and, due to their government-standard “Good Laboratory Practices” requirements, are very expensive.

This Tier, as designed, has the advantage that it can provide information on more human disease-focused data than governmental guideline studies at a fraction of the cost, and  can be adapted to enhance a guideline study to include EDC-sensitive endpoints, additional doses, and account for critical animal care issues that can have an impact outcomes, such as caging materials, light cycle, diet, handling, and strain differences.  Note That mammalian studies can and should be done with a TiPED collaborator experienced with both mammalian screening and risk assessment requirements.

Disadvantages:

Rodent testing is not high-throughput, requires labor-intensive monitoring, demands specialized skills to do properly, and is expensive.

A potential pitfall of this Tier is that rodents are sensitive to the laboratory environment, with husbandry, food, temperature, social environment, and other factors changing basic developmental and physiological processes. These tests must be conducted in partnership with an experienced TiPED collaborator once the compound of interest has been screened in at least one of the other tiers to obtain basic information about its potential EDC activity. It is absolutely essential that the person conducting the assays be trained and a confirmed expert on each aspect of the work.

Because we respect the importance of minimizing animal testing, appropriate statistical analyses such as power analysis must be conducted prior to performing any work. Additionally, teams of experts working together are the ideal scenario to collect tissues to minimize use of  animals. These caveats underscore the point that experienced TiPED partners are needed to design, conduct, and interpret results.

Fish and Amphibian Assays’ Strengths and Weaknesses

Strengths:

The advantages of the screens discussed here are that they are conducted in a physiologically intact vertebrate system.  The endocrine system is complex, and this tier captures some of this complexity.  Thus instead of testing specific cells or tissues, one can see chemical effects on a whole animal.

By testing effects on animal development during the earliest life stages, the probability of capturing an adverse effect is markedly heightened. Because fish reproduce in great number and develop quickly, one can do many assays on many individuals, thus giving a clearer result. Stages of development are much shorter than in mammals, and access to embryos for manipulation and observation is easier because they are not in a womb.

Another strength of the fish assays is that scientists have made significant progress in automating the assays, moving partially toward a version of high-throughput screening approaching the high throughput cell-based assays which might be deemed “medium-throughput assays.” This will lead to cheaper, faster and more accurate assays.

Limitations:

Disadvantages arise from the fact that, while we can learn a lot from them, fish and amphibia obviously are not mammals, and as such cannot provide all the answers that one might want about the potential human-relevant endocrine activity of a given compound.

Another disadvantage is that, while these assays can give an excellent overview of potential toxic effects, they are less good at explaining why these effects occur.  For the chemist who wants to know the specific mechanism by which a chemical is endocrine-active in order to re-design that chemical to make it benign, tests at the mammalian level, and/or specific tests at the cellular level, would be needed.

Cell-Process Assays’ Strengths and Weaknesses

Strengths:

Cell-Process assays capture endpoints that may be missed by the previous two tiers, for example nongenomic actions of nuclear hormone receptors. Therefore, they are valuable and could be performed in preference to computer based and target cell assays if budget is limited and broad answers are required. Assays in this tier are cell-process based; therefore, they can interrogate integrated pathways by measuring function and can be more sensitive for detecting endpoints than receptor specific assays. For example, BPA is a poor estrogen in receptor-based assays but a very strong estrogen in Cell-Process proliferation assays and in non-genomic signaling assays.

Cell-Process assays can provide a higher level analysis of compound action since many potential routes of, for example, estrogenicity can be interrogated in a single assay (nuclear estrogen receptor activity, membrane estrogen receptor activity, alterations in the levels of endogenous estrogens or of co-factors required for receptor activity). Therefore, the sensitivity of Cell-Process assays may be higher in some cases than target based assays in other tiers.

Limitations:

Cell-Process assays might not reveal complete details of the mechanism of action. For example, knowing that a chemical was estrogenic because it promoted proliferation of estrogen-dependent cancer cells would not necessarily reveal just how the chemical acted (i.e., which receptor it acted on and how).

Cell-Process assays are not currently available for all possible endocrine endpoints and are not a substitute for assays that measure the activity of chemicals, in vivo, on an integrated living system rather than isolated cells. Since not all possible cell types are represented, whole animal assays in the Fish/Amphibian and Mammalian tiers might well be required to capture all possible actions.

Additionally, only some cell-process assays are commercially available and we have not vetted the existing suppliers of assays. Technical care is required to successfully execute these assays, necessitating the development of robust assays that employ multiple positive and negative control chemicals (see TiPED Guiding Principles). While it is unlikely that chemicals positive in this Tier will be negative in whole animal tiers, it is possible that chemicals which are negative in this tier could have effects in whole animal tiers.

Amphibian

As non-amniotes (lacking egg shells or fetal membranes as embryos) without barriers to chemical contaminants, amphibians are highly susceptible to contaminant exposure and are thus exceptionally good indicators of environmental disturbance. Even as larvae and adults, their moist permeable skin provides easy access for chemical contaminants to cross.

Frogs are the preferred amphibian model.  Frogs show responses to thyroid hormones, androgens, estrogens, and corticoids, and biological markers that can detect disturbances in all four of these hormone classes have been defined and developed.

Selecting species for amphibian assays

One major benefit of the amphibian model is the dependence of metamorphosis on proper signaling of the pituitary and thyroid. Compounds that inhibit metamorphosis could do so by interfering with any aspect of thyroid hormone synthesis, transport, receptor binding, action or degradation. Similarly, compounds that stimulate or accelerate metamorphosis can act via multiple mechanisms.

To screen chemicals for androgen agonist/antagonist activity, several assays are available. Because exogenous androgens can sex-reverse some species of amphibian larvae, by monitoring sex ratio, androgen mimics can be detected. In addition, several androgen-dependent secondary sex characters can be assayed including laryngeal size, gular pouch development, and breeding glands[47,48]. There are also several behavioral assays available to examine androgen dependent reproductive behavior and functional assays that examine fertility in males[49].

Amphibians are also useful for screening chemicals that influence estrogen levels.

Finally, because corticoids affect growth, osmoregulation, and immune function, among other aspects of amphibian development, the effects of corticoid agonists/antagonists are more complicated to assay. Often, compounds that interfere with corticoids do so by increasing or inhibiting corticoid synthesis, which is easily monitored in vivo along with the assays described above. Importantly, mechanisms other than corticoid agonism/antagonism or changes in corticoid synthesis could explain many of the effects described here [51].