Drug toxicology and translational technology
Lecture 1
12/04/2021 Anika Nagelkerke
We are interested in the toxicological effects that we may find in humans. For that we need
some kind of predictive testing for toxicology. Animal models have a limited predictability for
humans and animal use is ethically more-and-more undesirable. There is a need for
alternative technologies that are more translational and are better for predicting the
toxicological effects in humans.
The 3 Rs:
- Replace → animal studies with ‘lower’ animals or non-animal animals.
- Reduce → animal studies to minimum required and necessary. Can you do the same
study with less animals?
- Refine → practices to minimise stress of study animals. So you need to comfort the
animals the most possible via e.g. cage enrichment (good conditions, food,
environment).
Toxicology/toxicity is the degree to which a substance (at a certain dose) can harm (part
of) an organism. This can be acute (immediate, high doses, minutes/hours/days → most
severe outcome is death) or chronic (gradual, sublethal/low dose, long-term → subtle
changes and cumulative damage which can also lead to death). Toxicity can be desired, for
example in cytotoxicity of cancer treatment in cancer cells (toxicity is the remedy). But it can
also be undesired, for example cytotoxicity in healthy cells due to cancer treatment (toxicity
leading to side effects).
1
,Translational technologies are technologies that enable the prediction of human
toxicology. For example idiosyncratic reactions, late effects, risk assessment (safe dose
levels) and drug selection.
- In vitro → cell or tissue cultures.
- Ex vivo → tissue or organ cultures.
- In silico → modelling.
Specificity of toxicity
If multiple tissues are working together they
form an organ. If organs work together e.g.
in the GI tract this is an organ system. So
there are different cells that make up tissues
that make up organs that make up organ
systems that make an organism. Adverse
effects can be seen on each level. If it acts
throughout the entire organism this is called
a systemic effect. But also single organs or
tissues can be affected → organ and tissue
specific effects can still lead to adverse
effect on the organism level. So the
organism is still at risk on dying. These
effects are highly dependent on the dose
that is given and on the route of
administration.
Homeostasis: maintenance of the body’s internal
steady state. If you have an imbalance by a stimulus
or a toxicant/drug, the change is detected by e.g.
receptors, then the input is sent via the afferent
pathway to the control centre. Then output is sent via
efferent pathway to the effector which will induce a
response to correct the imbalance. Homeostasis does
not involve keeping conditions static. It involves
keeping conditions within tightly regulated
physiological tolerance limits.
Homeostasis is a continuous turnover of cells. If you have an injury, rapidly a number of cells
can be damaged. This can, dependent on the severity, still be solved. E.g. in the brain
regeneration is limited while in the liver regeneration is quite high. If regeneration is not
possible, repair can be induced e.g. in wounds by the formation of a scar tissue. The
structure is changed but it is still functional. When the injury is so severe and cannot be
repaired it can result in death. For toxicants, the outcomes depend on the characteristics of
the cells, dose duration, organism (genetics/underlying disease) and environmental factors.
This is on a cellular level.
2
,Paracetamol: metabolism in the liver by UDP glucuronosyl transferase and sulfotransferase.
If these pathways are saturated, CYP2E1 can form NAPQI. This can be detoxified by
glutathione 5-transferase or bind covalently to SH groups which results in toxicity and cell
death. There is no life-threatening toxicity in the kidney after a high dose of paracetamol
because there is first-pass metabolism in the liver (the liver is exposed to more paracetamol)
and because there is less CYP2E1 in the kidney.
Some drugs have specific effects in certain organs and tissues. Causes for this differences in
organ and tissue uptake, biotransformation, bioconcentration, tissue-specific mechanisms
and the route of administration.
There are different mechanisms for specificity of toxicity.
1. Organ and tissue-specific mechanisms and effects → such as hepatotoxicity,
nephrotoxicity etc. All these organ systems have specific responses to toxicity. For
example, hepatotoxicity is a major issue in both pre-clinical and clinical development
and many drugs are withdrawn due to hepatotoxicity.
2. Developmental stage-specific mechanisms → whether a compound is e.g.
hepatotoxic or cardiotoxic depends on the characteristics of the tissue, dose duration,
route of administration, distribution, organism and environmental factors. We would
like to be able to predict these characteristics.
3. Species-specific mechanisms → all species differ in their expression of metabolic
enzymes, drug transporters, etc.
Embryotoxicity spans over both the organ and tissue-specific mechanisms and effects and
the developmental stage-specific mechanisms. Embryotoxicity is quite special. In a sense it
is specific for certain structures, but these embryonic stem cells have a high capacity for
repair in comparison to adult tissues. In a way this could also provide us important
information about repair which can be exploited in adult tissue.
Genotoxicity spans almost every single tissue and organ so is not limited to a specific organ
and tissue type. These cause damage to DNA, which is present everywhere in every
developmental stage and every organ and tissue. The FDA considers this the most
dangerous type of toxicity, because it can affect the entire body.
3
, Diclofenac is used by farmers to keep their animals safe. But then vultures (birds) eat the
carcasses of the dead cattle and thereby ingest diclofenac. They were not able to metabolise
this, leading to kidney failure and visceral gout. So here you see that diclofenac has species-
specific effects.
The basics of cell culture
After drug exposure we would like to predict the effects on organism level. But we need
something to study this with → organs and organ systems. But these are not yet as far
developed that they can actually be used and you still need an organism to get the organs
from. So cells and tissues are the way to go.
How to make a cell culture: we can grow all kinds of cell types outside of our body. The
way that we get cells into culture starts from tissue, so we still need to use animals or
humans. So you have the tissue/organ with the cell type of interest. This is dissected to
remove damaged and necrotic parts. You keep the healthy tissue which you can
disaggregate using mechanical or enzymatic disaggregation. These explants are dispersed
in medium followed by incubation and growth of the culture. Then you can separate and
purify the cell type of interest by selective media or via marker selection. Then you end up
with cells stuck to the plastic of the petri dish. They can be very demanding. It is possible to
give them a VELCRO which is an extracellular matrix (ECM) that the cells like to stick to. We
also give them lots of food via a cell culture medium which consists of a lot of sugar and
nutrients to keep them happy. Cells are moving and dividing inside the petri dish. If you want
to make an in vitro model that approaches humans we always need cells.
In drug screening, you can add a drug to the cell culture medium, the drug then reaches the
cells to exert an effect. So for initial drug screening purposes to see whether they are toxic or
not and whether they exert the wanted effect, such methods are quite useful.
How representative is a petri dish for human
physiology? Petri dishes contain monolayer cultures
stuck to the surface → 2D. We are able to form 3D
models. For this you need structural support via a
scaffolding ECM. So you need a material that beats
the gravity. This has consequences for how cells
behave.
4
Lecture 1
12/04/2021 Anika Nagelkerke
We are interested in the toxicological effects that we may find in humans. For that we need
some kind of predictive testing for toxicology. Animal models have a limited predictability for
humans and animal use is ethically more-and-more undesirable. There is a need for
alternative technologies that are more translational and are better for predicting the
toxicological effects in humans.
The 3 Rs:
- Replace → animal studies with ‘lower’ animals or non-animal animals.
- Reduce → animal studies to minimum required and necessary. Can you do the same
study with less animals?
- Refine → practices to minimise stress of study animals. So you need to comfort the
animals the most possible via e.g. cage enrichment (good conditions, food,
environment).
Toxicology/toxicity is the degree to which a substance (at a certain dose) can harm (part
of) an organism. This can be acute (immediate, high doses, minutes/hours/days → most
severe outcome is death) or chronic (gradual, sublethal/low dose, long-term → subtle
changes and cumulative damage which can also lead to death). Toxicity can be desired, for
example in cytotoxicity of cancer treatment in cancer cells (toxicity is the remedy). But it can
also be undesired, for example cytotoxicity in healthy cells due to cancer treatment (toxicity
leading to side effects).
1
,Translational technologies are technologies that enable the prediction of human
toxicology. For example idiosyncratic reactions, late effects, risk assessment (safe dose
levels) and drug selection.
- In vitro → cell or tissue cultures.
- Ex vivo → tissue or organ cultures.
- In silico → modelling.
Specificity of toxicity
If multiple tissues are working together they
form an organ. If organs work together e.g.
in the GI tract this is an organ system. So
there are different cells that make up tissues
that make up organs that make up organ
systems that make an organism. Adverse
effects can be seen on each level. If it acts
throughout the entire organism this is called
a systemic effect. But also single organs or
tissues can be affected → organ and tissue
specific effects can still lead to adverse
effect on the organism level. So the
organism is still at risk on dying. These
effects are highly dependent on the dose
that is given and on the route of
administration.
Homeostasis: maintenance of the body’s internal
steady state. If you have an imbalance by a stimulus
or a toxicant/drug, the change is detected by e.g.
receptors, then the input is sent via the afferent
pathway to the control centre. Then output is sent via
efferent pathway to the effector which will induce a
response to correct the imbalance. Homeostasis does
not involve keeping conditions static. It involves
keeping conditions within tightly regulated
physiological tolerance limits.
Homeostasis is a continuous turnover of cells. If you have an injury, rapidly a number of cells
can be damaged. This can, dependent on the severity, still be solved. E.g. in the brain
regeneration is limited while in the liver regeneration is quite high. If regeneration is not
possible, repair can be induced e.g. in wounds by the formation of a scar tissue. The
structure is changed but it is still functional. When the injury is so severe and cannot be
repaired it can result in death. For toxicants, the outcomes depend on the characteristics of
the cells, dose duration, organism (genetics/underlying disease) and environmental factors.
This is on a cellular level.
2
,Paracetamol: metabolism in the liver by UDP glucuronosyl transferase and sulfotransferase.
If these pathways are saturated, CYP2E1 can form NAPQI. This can be detoxified by
glutathione 5-transferase or bind covalently to SH groups which results in toxicity and cell
death. There is no life-threatening toxicity in the kidney after a high dose of paracetamol
because there is first-pass metabolism in the liver (the liver is exposed to more paracetamol)
and because there is less CYP2E1 in the kidney.
Some drugs have specific effects in certain organs and tissues. Causes for this differences in
organ and tissue uptake, biotransformation, bioconcentration, tissue-specific mechanisms
and the route of administration.
There are different mechanisms for specificity of toxicity.
1. Organ and tissue-specific mechanisms and effects → such as hepatotoxicity,
nephrotoxicity etc. All these organ systems have specific responses to toxicity. For
example, hepatotoxicity is a major issue in both pre-clinical and clinical development
and many drugs are withdrawn due to hepatotoxicity.
2. Developmental stage-specific mechanisms → whether a compound is e.g.
hepatotoxic or cardiotoxic depends on the characteristics of the tissue, dose duration,
route of administration, distribution, organism and environmental factors. We would
like to be able to predict these characteristics.
3. Species-specific mechanisms → all species differ in their expression of metabolic
enzymes, drug transporters, etc.
Embryotoxicity spans over both the organ and tissue-specific mechanisms and effects and
the developmental stage-specific mechanisms. Embryotoxicity is quite special. In a sense it
is specific for certain structures, but these embryonic stem cells have a high capacity for
repair in comparison to adult tissues. In a way this could also provide us important
information about repair which can be exploited in adult tissue.
Genotoxicity spans almost every single tissue and organ so is not limited to a specific organ
and tissue type. These cause damage to DNA, which is present everywhere in every
developmental stage and every organ and tissue. The FDA considers this the most
dangerous type of toxicity, because it can affect the entire body.
3
, Diclofenac is used by farmers to keep their animals safe. But then vultures (birds) eat the
carcasses of the dead cattle and thereby ingest diclofenac. They were not able to metabolise
this, leading to kidney failure and visceral gout. So here you see that diclofenac has species-
specific effects.
The basics of cell culture
After drug exposure we would like to predict the effects on organism level. But we need
something to study this with → organs and organ systems. But these are not yet as far
developed that they can actually be used and you still need an organism to get the organs
from. So cells and tissues are the way to go.
How to make a cell culture: we can grow all kinds of cell types outside of our body. The
way that we get cells into culture starts from tissue, so we still need to use animals or
humans. So you have the tissue/organ with the cell type of interest. This is dissected to
remove damaged and necrotic parts. You keep the healthy tissue which you can
disaggregate using mechanical or enzymatic disaggregation. These explants are dispersed
in medium followed by incubation and growth of the culture. Then you can separate and
purify the cell type of interest by selective media or via marker selection. Then you end up
with cells stuck to the plastic of the petri dish. They can be very demanding. It is possible to
give them a VELCRO which is an extracellular matrix (ECM) that the cells like to stick to. We
also give them lots of food via a cell culture medium which consists of a lot of sugar and
nutrients to keep them happy. Cells are moving and dividing inside the petri dish. If you want
to make an in vitro model that approaches humans we always need cells.
In drug screening, you can add a drug to the cell culture medium, the drug then reaches the
cells to exert an effect. So for initial drug screening purposes to see whether they are toxic or
not and whether they exert the wanted effect, such methods are quite useful.
How representative is a petri dish for human
physiology? Petri dishes contain monolayer cultures
stuck to the surface → 2D. We are able to form 3D
models. For this you need structural support via a
scaffolding ECM. So you need a material that beats
the gravity. This has consequences for how cells
behave.
4
