In this section, we'll talk about adverse outcome pathways relevant to toxicity pathways and relevant to the Endocrine Disruptor Screening Program. The Endocrine Disruptor Screening and Testing Program takes a pathway-based approach. We're focused on looking at three endocrine pathways: the estrogen, the androgen, and thyroid pathways. We're interested in looking at the interactions of chemicals or toxicants as they find their molecular targets in cells, as they drive a cellular response in an organ, as that drives an organismal response and eventually, in the case of wildlife and lead to population changes, adverse outcomes; in either individuals or in populations. And there's this connection linking key events from the molecular interaction to the adversity seen in individuals and in populations. This is the pathway-based approach to Endocrine Screening and Testing, when generally it's a pathway-based approach to identifying toxicity, to identifying adverse outcomes, and to identifying adverse outcome pathways; linking chemical exposures to adversity. In the Endocrine Screening and Testing Program, where as I've mentioned, looking at three different pathways, the estrogen pathway indicated here E+ E- as an estrogen actives or estrogenic chemicals in anti estrogenic or estrogen blocking or inhibitory chemicals. Androgen and anti androgen A+ and A-. And thyroid pathway, Hypothalamic Pituitary Thyroid axis, HPT in the bottom row of this table. And you can see that all of the Tier 1 and Tier 2 type assays, which are the foundation for how we go about screening and testing, and any alternatives we develop, can be organized along these three different endocrine pathways: estrogen, androgen, and thyroid. And can also be organized starting from the left, from the molecular interactions and cellular interactions leading into the organismal and population adverse outcomes, particularly in Tier 2, where we are identifying dose response and adversity in those extended one generation and multigeneration reproductive and developmental tests. And so, there's an under girding of the entire endocrine screening and testing program related to these three pathways: estrogen, androgen, and thyroid. Relating to toxicity pathways and adverse outcome pathways. Adverse outcome pathways link as I've mentioned, the interaction of a molecule with a cellular or biological target, a molecular target to an adverse outcome. In the case of endocrine pathways often to adverse developmental or reproductive outcome. So an example, would be aromatase inhibition, disrupting normal egg production, and leading to population changes in fish. So an example here would be fadrozole and aromatase inhibitor which could reduce estradiol synthesis which reduce circulating Estradiol concentrations, which could reduce vitellogenin production, which could reduce spawning, which could end up in a reduction in population of a particular fish species. So this would be an example of an adverse outcome pathway leading from molecular interaction. In this case, fadrozole, aromatase enzyme all the way through to an adverse outcome of reduced population of that particular species. AOPs can integrate a range of different types of laboratory tests, non-animal test, to animal tests, spanning this molecular to population level of impact. So starting at the bottom of this diagram, you have that target of molecular interaction. Remember the fadrozole example in the previous slide interacting with the aromatase enzyme, could be any molecule interacting with any molecular target that then link to an organelle or cellular response that could lead to a tissue response or organ response to an organismal response. Perhaps, decrease spawning as in the fadrozole example. And that potentially, could in wildlife populations lead to population decline. And in humans, we would be particularly sensitive to individual responses. So I've mentioned that there's underlying connections. The endocrine screening and testing or endocrine disruptor screening program with these biological pathways and these adverse outcome pathway concepts. For estrogen, the adverse outcome pathway is identified through a network of a variety of different end points that are measured from high throughput screening and in silico assays looking at estrogen receptor activity through that tier one battery, the collection of different endpoints indicated in this diagram. Different key events leading from the left to the right or top to bottom, from toxicant and molecular interactions to a series of key events. And eventually, in tier two, impacts and fertility fecundity and normal development and general health of offspring. So there's a network of endpoints spanning a number of different adverse outcome pathways, relating to the broader estrogen pathway as indicated in this diagram. Within the program, we're using adverse outcome pathways to refine and develop integrated approaches to testing and assessment. So in the endocrine disruptor screening program, we're continuing to hone these concepts, similar to what I just showed you for the estrogen pathway. To link adverse outcome pathways to the screens and tests that we use in the program to identify and predict potential effects in humans and wildlife. And to develop improved testing and assessment methodologies. In the case of the Endocrine Disruptor Screening Program, we're using combinations of high throughput screening assays from ToxCast as indicated in this diagram, that span that molecular and to cellular range of responses in combination with Tier 1 type of assays, ranging from cellular to organismal responses. And Tier 2 assays. Again, looking at an organismal responses that are direct measurements of adversity and allows to determine the dose response for that adversity that is linked back through the integrated approach to the Tier 1 and high throughput screening or ToxCast assays. So linkage, if you will, exists through these pathways and through these key related key events in these pathways, linking adversity back to molecular interactions and endocrine bile activity. Here's a more diagrammatic form of these same concepts, starting from left to right with the molecular interactions. The molecular initiating event, MIE, leading to a series of key events resulting in adverse outcome. So that would be a very simple linear adverse outcome pathway. The toxicity pathway would be a subset of that, linking some specific molecular interactions with a subset of key events that are known to be related to either one or more adverse outcome pathways. And in the case of the estrogen receptor model, you could have data linking estrogen receptor activity and a subset of key events in a predictive model. So here's the example of the specific adverse outcome pathway. One example of ER agonism leading to altered development up at the top. And that a subset of those molecular interactions and key events linking ER agonism, increased ER activation and increased uterine weight in the toxicity pathway that is a subset of the adverse outcome pathway or collection of potential adverse outcome pathways. And that is predicted or an alternative exists using the estrogen receptor model to predict not only estrogen receptor agonism but also the increased uterine weight. So that's a prediction, if you will, from the ER model for that in vivo portion of the toxicity pathway. This is an abstraction then going back to the potential for multiple molecular initiating events leading to an adverse outcome pathway. And the potential for multiple models leading to those adverse outcomes. And representing a portion of the adverse outcome pathway in a relevant toxicity pathway, so here in the abstract model one and model two. And in this slide, the specific example of the estrogen receptor model and the steroidogenesis model dividing two different entryways if you will in that pathway leading to adversity and representing the toxicity pathway where estrogen receptor agonism or altered steroidogenesis or both activities could be indicate and predict the risk of endocrine disruption for any particular chemical or chemicals that either humans or wildlife are exposed to. And again, this could go on with greater and greater complexity. More and more models replicating or predicting greater portions of the toxicity pathway eventually perhaps an entire adverse outcome pathway or a network of adverse outcome pathways relevant to endocrine pathways: estrogen, androgen, thyroid or nonendocrine pathways and other types of adversity. This concludes the section on adverse outcome pathways and toxicity pathways. And we'll now move on to exposure-based chemical prioritization.
