This course familiarizes students with the novel concepts being used to revamp regulatory toxicology in response to a breakthrough National Research Council Report “Toxicity Testing in the 21st Century: A Vision and a Strategy.” We present the latest developments in the field of toxicology—the shift from animal testing toward human relevant, high content, high-throughput integrative testing strategies. Active programs from EPA, NIH, and the global scientific community illustrate the dynamics of safety sciences.
The Second Report: Toxicity Testing in the 21st Century: A Vision and A Strategy Vision and Its Components
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Из курса от партнера Johns Hopkins University
Toxicology 21: Scientific Applications
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Module 1
This model will give you a broad introduction to the Toxicology 21st century (Tox21c) field and familiarize you with the main document, which summarize the main ideas and principles of Tox21c.
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Lena SmirnovaResearch Associate
Center for Alternatives to Animal Testing
Thomas HartungProfessor
Environmental Health and Engineering
Welcome back to the lecture on the NRC report,
Toxicity Testing in the 21st Century, A Vision and A Strategy.
In this section,
section C of the lecture, we're going to talk about the second report.
Toxicity Testing in the 21st Century, a Vision and a Strategy.
And we're going to talk about the vision and its components.
The second report was comprised of six chapters.
An introduction, the vision, components of the vision, tools and
technologies, developing the science base.
And assays to implement the vision.
And then prerequisites for implementing the vision in regulatory contexts.
The committee envisioned a new toxicity-testing system that evaluates
biologically significant perturbations in key toxicity pathways.
Pathways is a key element of this new vision.
By using new methods in computational biology, and
a comprehensive array of in vitro tests based on human biology.
So if you read that carefully a couple times,
you will see that it's really a paradigm shift in the approach to toxicity testing.
And this would provide the opportunity for
broad coverage of chemicals, chemical mixtures, and outcomes in life stages.
It would reduce the cost of testing and also the time it takes to test.
It would use fewer animals and cause minimal suffering in the animals used, and
would develop a more robust scientific basis for
assessing health effects of environmental agents.
Now, the committee considered four options for future toxicity testing strategies.
Option I is the standard in vivo toxicity tests,
based on animal biology and high doses, the low throughput, and
it's expensive, time consuming, it uses large numbers of animals.
And it's based on the apical endpoints that I mentioned previously in another
section of this lecture.
So the first report pretty much reviewed the tests and
the issues associated with Option I.
In Option II, the committee thought about tiered in vivo tests.
In vivo is tests still using animals.
It would be based on animal biology and it would still have to use high doses.
But there could be improved throughput because down at the bottom as I've circled
a sum screening would be done, using computational and in vitro approaches.
And this would provide more flexibility than the current methods.
In other words, chemicals could be screened using computational methods,
determining their structure, activity, properties, and
then some in vitro approaches.
And you'll see, when you hear the talk on environmental estrogens,
that some of this occurs in the endocrine disruptor screening program.
It can reduce costs and also time.
It can result in fewer numbers of animals but it's still based on apical endpoints.
Option III that the committee considered was a combination of in vitro and in vivo,
primarily based on human biology because the in vitro would be mainly
human cells and it would also incorporate some human epidemiology.
One could use a broad range of doses.
When using cells in culture, you can use many, many doses,
expose the cells to many doses.
There would be opportunity for high and medium throughput.
In other words, when using cells in culture,
there are ways in which one can do assays that can be done using robotics.
It would be less expensive and less time consuming, and use substantially fewer
animals because animal tests would only be done in certain circumstances.
And it would be based on perturbations of critical cellular processes.
And this comes back to our concept of pathways of toxicity.
And it would also use screening via computational approaches.
Possible limited animal studies that focus on mechanism and metabolism.
One of the critical aspects of in vitro approaches, or
cell culture approaches, is that many times the cells in culture don't have
the ability to metabolize the chemical in a way that occurs in vivo.
And so one either has to know all the metabolites and
test them in a culture, or go to animal studies.
So that when the animal's exposed metabolism of the chemical does occur, and
if there's toxicity, the metabolites may be contributing to that.
And finally, Option IV was almost totally in vitro, primarily human biology.
A broad range of doses, as I indicated, can be used.
Can be very high throughput.
It's less expensive, less time consuming, and would use virtually no animals.
It's based on perturbations of critical cellular responses.
Again, these pathways of toxicity.
And screening can be done Using computational approaches.
So you'll see that where the committee ended up was mainly with Option IV.
But a little bit of Option III involved.
The big thing here is that this is kind of revolutionary.
It's really a shifting away from the normal approach to toxicity testing.
To one that involves virtually no animal use.
There are a number of issues that are related to this and
it's certainly not going to happen overnight.
The committee thought, well maybe 10 to 20 years before
the vision of the committee will be able to be fully implemented.
And there will be things in the in vitro systems that are just
at this point hard to think about how they would be tested.
For instance, novel agents like nanoparticles or
physical agents like biomedical implants that are used also have to be tested.
And how to test them with in vitro systems is
at this particular point not immediately apparent.
But there is a shift away from apical endpoints, and whole animal studies.
So the new paradigm is activation of toxicity pathways, and
that's diagrammed on this slide.
So, first of all, what's a toxicity pathway?
Well, I've defined it down at the bottom of the slide.
It's a cellular response pathway that, when sufficiently perturbed,
is expected to result in an adverse health effect.
So if you look at this diagram, and start at the top, there's an exposure,
there's a tissue dose, there's a biologic interaction.
This could be with the parent compound or
likely with a metabolite of the parent compound or chemical.
And then theres a perturbation.
And so, the horizontal line represents a biological process and
there are biologic inputs and there's a normal biologic function.
And so, if there's a perturbation in this pathway,
then there could be some early cellular changes.
In other words, a biochemical pathway now is, let's say, it's inhibited.
And this results in some early cellular changes.
But cells have the ability to adapt to altered homeostasis,
if you will, changes in any particular pathway, and return to normal.
And so, if an early cellular change is reversible through an adaptive response,
then normal biologic function would still be maintained.
However, if the perturbation that results from the chemical persists,
or if the perturbation is grave enough, then cell injury can result.
And this could, at the tissue level, organ level, and
finally organismal level cause morbidity or mortality.
It could cause an adverse health effect.
So the new paradigm is really based on identifying toxicity pathways
whose alteration could result in injury and then adverse health outcomes.
So, what are some toxicity response pathways?
Will they include hormone-responses?
And again, you'll hear more about that in the lecture on endocrine disruptors.
DNA damage, agents that caused DNA damage can have adverse effects on the cells.
And there are a number of pathways that resolved in the repair of the damage.
And so agents that actually interfere with those pathways could enhance
the DNA damage and the results from that damage.
Hypo-osmolarity, some chemicals could cause hypo-osmolarity of cells and
this could result in adverse effects.
There are a number of receptors that are listed there and
responses from these receptors can also result in adverse effects.
There's the Nrf2 oxidative stress pathway and they're also heat-shock proteins.
You don't have to know the details of these pathways but I present these
examples to show you that we already know that there are a number of pathways.
That can be monitored using high-throughput robotic type approaches in
cultured cells.
That when they are altered could result in adverse effects on
the cells that could translate to toxicity in tissues and organs and
cause adverse health effects if the organ is mid-level.
Now, let's just take a look at one complex pathway and that is estrogens.
So estrogens can exert effect on the cells through a variety of mechanisms.
And you see on the right-hand side of this chart,
at the far right, there's an arrow going from estrogens to estrogen receptors.
So estrogen action in the cell occurs predominately through estrogen receptors.
And these receptors can then affect through both mechanisms that occur
in the nucleus of the cell, a genomic.
Where they alter the transcription of genes through receptors
that are referred to as non-genomic that exists in the cytoplasm of the cell.
That can affect second messenger systems in the cell and
receptors also go to the mitochondria of the cell.
And altered gene transcription in the mitochondria.
And all these can come together to alter gene expression.
And an alter gene expression in response to estrogens can cause
increased cell proliferation, increased cell growth, or
decreased cell death which is referred to as apoptosis.
And if this occurs in inappropriate ways, then it can result in
adverse affects at the tissue, organ, and ultimately organismal level.
Estrogens can also cause epigenetic effects which can alter
gene transcription in the nucleus.
So these are complex pathways but
they can be assessed using high-throughput methods in cultured cells.
On the left-hand side, I also indicate here that estrogens are metabolized.
And so you can see that there are a number of oxidative metabolites that
result from estrogen metabolism.
There are the catechols and the quinones.
The quinones are very reactive metabolites and
they cause oxidative DNA damage affecting specific bases in the DNA.
And so one has to keep track of whether or not the estrogens are metabolized,
and some cultural systems may not be the best for doing this.
Some of this has to be done in higher levels of organization like
in an animal or different, maybe in vitro methods or
structure activity analysis can indicate that.
Well, this particular estrogenic compound would be metabolized in this way.
And so you would then pick maybe synthesize that metabolite and
test it in the cell culture system to see if it disrupted any of
the estrogen-signaling pathways.
In addition, estrogens are made.
They're made from androgens in the human body.
And so one has to also think about whether or not an environmental agent
disrupts the formation of estrogens, the biosynthesis of estrogens.
So this is a very complex process and the in vitro systems will
have to be designed to be able to pick up perturbations in any of these pathways
that may be caused by an environmental chemical.
Another example is this NRF-2 Response Pathway.
This is an antioxidant response pathway.
And you see on the right-hand side, oxidants can enter the cell and
they interact with this complex that shown in the lower right hand side of the cell.
Which is complex of Nrf2 with this other protein Keap1, and Cul3.
And when that occurs, then NrF2 is released from this complex,
as you see up toward the top.
It's activated and it enters a nucleus and
turns on antioxidant stress response genes.
Among those are also phase two detoxifying enzymes.
So an environmental agent that interferes with this pathway
could actually cause damage within a cell.
So this is just a couple of examples of specific pathways that can be
accessed through high-throughput screening in cultured cells.
So the committee spent a long time
coming up with a diagram that will kind of capture its vision.
I remember those discussions and
there were many diagrams that members of the committee proposed.
And finally we all agreed on this diagram which captures this vision.
So if you look at the top, there's chemical characterization.
This is a very important component as more and
more data becomes available than there are structure activity.
And other computational tools that allow us to classify chemicals
according to their structure and potential for metabolism.
And so the first step is, of course, chemical characterization.
You don't need to do any testing before you
actually do that kind of in silico analysis in the chemical.
And then there's testing, a whole series of toxicity pathway tests.
This would be done again through a high-throughput robotic processes.
And there will be occasions where targeted testing in animals maybe needed.
Either because the nature of the chemical itself or because additional information
is needed, perhaps the role of metabolites in causing an adverse effect.
But these would be very focused tests and
perhaps not needing to use large numbers of animals when they have to be done.
And there's also important across the board when testing is dose response and
extrapolation modeling.
I'll talk a little bit more about that in a few minutes because you can see
that if we're exposing cells in culture.
How do we know that the concentrations that the cells have been exposed to
are at all similar to the concentrations in tissues of a human that had been
exposed to that chemical through the environment?
And wrapped around this whole thing, first are population-based and exposure data.
Many of these chemicals are already in commercial use.
We have population data on exposures to some of them and
sometimes associations of adverse outcomes with this exposure data.
This kind of data needs to be incorporated in to the vision for
the future of toxicity testing.
And we also have to take in context the risk context.
In other words, who is exposed and what level do we think they're exposed to?
We may need only certain types of tests based on how widespread
the exposure is going to be and who might be the exposed individual.
I thought when we finally came up with this really it
captures everything that we are talking about.
Now let’s explore this a little bit.
I'm not going to go integrate detail here.
You can study this as you go through these slides.
But you see when we look at chemical characterization.
As I indicated we have to compile data on the physical and chemical properties and
use this knowledge in our decisions as to what needs to be done next.
We should be able to predict the properties and
characteristics using computational tools.
And we can answer key questions concerning the compound stability potential for
human exposure bioaccumulation, which is an extremely important issue.
Is this chemical going to bioaccumulate up the food chain and
bioaccumulate in humans that are exposed to it, etc.
So chemical characterization is critical.
And when we talk about toxicity testing,
the pathway testing, we have to evaluate the perturbations in these pathways.
We have to discover these pathways.
There's a lot of research that has to be done to discover
what are the critical toxicity pathways in various tissues, liver,
heart tissue, brain tissue, etc rather than using apical end points.
Once we know that these pathways are important, then we will come up with
means in which we can assess end points that indicate that the pathway
has been altered, and again through high throughput robotic driven processes.
We often emphasize this high throughput approach because that's what's going to
allow us to do multiple doses and also screen many, many compounds.
Remember 80,000 chemicals out there, very few of which have been thoroughly,
independently tested.
And then there can be some targeted testing as I've indicated before,
to evaluate metabolites and asses target tissues,
where we really aren't able yet to do a good job, en vitro or
where there may be some novel compounds that we have to test in an animal.
The committee envisioned that there will be a lot of animal testing that will
continue until the pathway approach begins to become more robust.
And as that happens, then the use of animal testing will decline.
And as I indicated previously, this isn't going to happen overnight.
It may take ten to 20 years before there is a total shift to the use of this
implementation and realization really, of this new vision.
In the dose responsive extrapolation modeling,
there will be the development of these empirical dose response models
that are based on the data from the in vitro, mechanistically based assays.
There would be physiologically based pharmacokinetic models that will equate
the tissue-media concentrations from toxicity tests with tissue doses that
are experienced in humans that are exposed to these chemicals in the environment.
And the goal is that these dose-response models for the pathways where we reliably
predict concentrations expected to cause and measure precursor effect responses.
And the PBPK and toxicity pathway models will identify biomarkers of
susceptibility for sensitive sub-populations, so that from this in
vitro mechanistic approach using the pathways, we may be able to identify
biomarkers that actually can be translated to the human population and used.
They may allow us to determine the susceptibility
of sensitive human sub-populations.
With regard to population and exposure data, I'm not going to read through all
of this, but in essence, we can do a better job of using population data to
both the association of adverse effects with exposures as well as examples and
incorporate where exposure has been assessed.
That can help us drive out in vitro dose response studies.
And, finally, the risk contexts, when we evaluate new environmental agents,
we want to really determine what are the potential environmental
contributors to a specific disease?
And we want to evaluate the relative risks associated with these
environmental agents.
But with regard to the risk context, who is going to be exposed,
at what level, and for how long?
And this may determine the extent to which we actually do a test.
Some tests can be reduced considerably and be very focused, so
that we don't have to run through just a given set of tests.
There can be flexibility as to when we have enough data and
we can stop further testing.
So the toxicity testing and risk assessment vision that we're thinking of
is consistent with the original risk assessment paradigm that was originally
put forth by a National Research Council report in 1983.
It really comprises the red book that's used by the FDA.
It involves, as you can see on the chart, hazard identification but
a whole new approach to hazard identification
that involves chemical characterization and mode of action.
The mode of action is assessed in biological systems.
We look at perturbations of these pathways of toxicity.
How are the pathways effected and at what dose levels are they effected?
And this comes together to give us a dose response analysis for
perturbation of these pathways.
And then this feeds into calibrating the in vitro and human dosimetry.
How do we connect that in vitro exposure level to what actually
may be occurring in humans that are exposed through the environment?
And this involves the PBPK modeling and
dose response modeling that has to be developed.
It's in the process of being developed.
But much more work needs to be done in that way.
We want to incorporate population based studies, both on human exposure data as
well as the association of adverse effects that we pick up in epidemiology study.
And all of this will come together to establish a human exposure guidelines
that would be based on risk avoidance.
So this concludes this section of the lecture.
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