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The role of biokinetics in in vitro tests and the interpretation of results
Emanuela Testai
Istituto Superiore di Sanità
Department of Environment and Primary Prevention Mechanisms of Toxicity Unit
Rome-Italy [email protected]
Rome 17.12.2012
International Symposium on
Alternative in vitro methods to characterize the role of EAS in hormone-targeted tissues
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Alternative in vitro methods to characterize the role of EAS
The International Programme for
Chemical Safety (2002) has
established the following definition for
endocrine disrupters:
Endocrine disrupters are exogenous
substances or mixtures that alter
function(s) of the endocrine system
and consequently cause adverse
health effects in an intact organism,
or its progeny, or (sub)populations.
US EPA and IPCS do not consider endocrine disruption to be an adverse
effect per se, but rather to be a mode or mechanism of action potentially
leading to other outcomes, i.e. carcinogenic, reproductive/developmental
effects, routinely considered in reaching regulatory decisions.
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There are at least two clear requirements for a substance to be
defined as an ED: the demonstration of an adverse effect and of an
endocrine disruption mode-of-action (biological plausibility).
Alternative in vitro methods to characterize the role of EAS
Difference between endocrine modulation and endocrine disruption.
Many adaptive, compensatory, and physiologically normal/necessary
processes result in measurable endocrine changes.
It is only when these natural mechanisms are affected to such a degree that
adverse effects are induced that ED occurs.
Exogenous chemicals in order to affect the endocrine system must act against
the background of circulating levels of endogenous hormones, which are
usually much more potent than any ED (the potency issue).
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In vitro studies can be used to study the MoA and for priority testing
based on hazard.
For the time being their use for risk assessment purposes is limited due to
difficulties in carrying out quantitative in vitro to in vivo extrapolation.
Need of translating information from the cell level, to organs and subsequently
to organisms and to distinguish between adaption vs. adversity, likely
identifying actual in vitro marker of adversity.
‘Omics’ techniques are producing a bulk of information, but before we can
quantitatively use it more knowledge is need for a correct interpretation.
Alternative in vitro methods to characterize the role of EAS
Lack of information on actual exposure of cell: in this respect in vitro
biokinetics data providing the actual level of cell exposure producing an in
vitro observed effects can improve the in vitro-in vivo extrapolation.
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The knowledge of the bioavailability of a given compound by the relevant
uptake routes should represent the starting point for any toxicological testing.
In vivo the actual internal dose reaching the target is the more relevant
parameter in evaluating exposure and in the quantitative risk assessment.
When developing testing strategies, kinetics is considered the crucial body of
information for the design and performance of toxicological tests and for
toxicity data interpretation.
This consideration applies also to alternative/non
animal testing strategy
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Alternative in vitro methods to characterize the role of EAS
Biokinetics processes have
been evoked to explain the in
vitro/in vivodifferences
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In vitro biokinetics
Identification of actual cellular exposure (peak concentrations, AUC,
parent vs metabolites)
The actual intracellular concentration may greatly differ from the
nominal applied concentrations due to
altered bioavailability : interactions with the medium components, the
plate, the cell itself, evaporation, chemical instability (abiotic
processes).
physiological cellular processes : mechanism of transport across the
membranes, biotransformation, bioaccumulation.
In vitro the nominal applied concentration rather than the actual level
of cell exposure is usually associated to the observed effects.
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Alternative in vitro methods to characterize the role of EAS
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Cells
Test Item
Plastic binding
Evaporation
Protein binding
Uptake
Free Concentration in the medium
Target
Metabolism Free
Concentration
Characterization of the cell model
Passive/Active
(Transporters)
Alternative in vitro methods to characterize the role of EAS
In vitro biokinetics
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WP3: Non animal-based models for in vitro kinetics and human
kinetic prediction
Predict-IV—Profiling the toxicity of new drugs: a non
animal-based approach integrating toxicodynamics and
biokinetics
The ultimate goal is to contribute to the derivation of NOEC values (relatively to drug safety) in model systems based on human cells representative of in vivo target organs, from which it would be possible to extrapolate the corresponding in vivo dose.
In repeated treatments for prolonged time of exposure the uncertainty about the actual level of exposure of cells in vitro is greatly enhanced (possibility of induction/inhibition phenomena, cumulative toxicity).
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Alternative in vitro methods to characterize the role of EAS
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D0 D13
comparison
Daily treatment
5 time points 5 time points
Cellule
Tempo
Co
ncen
trazio
ne
0 1 2 3 4
0
10
20
30
40
50
20 24
Cellule
Seeding
Cellule
Tempo
Co
ncen
trazio
ne
0 1 2 3 4
0
10
20
30
40
50
20 24
Cellule
First day/first dose) Last dose
Sample collection and transport according to specific SOP
Extration from biological matrix according to specific SOP
Quantitative analysis
Alternative in vitro methods to characterize the role of EAS
Concentrations
Primary rat hepatocytes (PRH)
Primary human hepatocytes(PHH)
HepaRG
HD= TC10
LD= 1/10 di TC10
Human renal cells (RPTEC/TERT1)
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Human kidney cells (RPTEC/TERT1): CSA LD - HD: 5-15 µM
cells
medium
LD
cells
HD
Alternative in vitro methods to characterize the role of EAS
Adsorption to the plastic in this system not relevant
High potential for bioaccumulation
Kinetic of intracellular conc
and in the medium during 24
hrs very low metabolic
competence
Wilmes A., et al. J. Proteomics 79, 180-194 (2013)
11 Wilmes A., et al. J. Proteomics 79, 180-194 (2013)
⃝ LD ∆ HD CsA
Biokinetic model: (A) CsA supernatant concentration
(B)CSA Intracellular concentration
Alternative in vitro methods to characterize the role of EAS
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In the case of endocrine active substances a strong debate is going on about the ‘low dose hypothesis’, according to which “low dose effects”, which are not present at higher doses may display a non-monotonic dose-response (NMDR). Therefore for those given effect, a simple monotonic extrapolation from high to low doses during risk assessment of those substances is no more valid.
environmentally-relevant doses
doses in the range of typical human exposure
doses below those used in traditional toxicological studies
doses below the presumed NO(A)EL or BMDL derived by testing
Which is the actual definition of low doses?
Can kinetics information help in explaining controversial aspects in the area of ED ?
Alternative in vitro methods to characterize the role of EAS
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Starting from Paracelsus statement
All substances are poisons. It’s the dose that makes the poison
the paradigm in toxicology and risk assessment is that the individual
response of an organism to a chemical increases/decreases proportionally
to the exposure (dose).
It is generally accepted that for most
chemicals (with no genotoxic potential)
there is a threshold dose below which
there is no adverse effect.
This gives rise to a monotonic dose-response relationship
In monotonic responses the effect either increases or decreases
over the full dose range tested.
Alternative in vitro methods to characterize the role of EAS
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The possibility exists that non monotonic dose-response relationship
occurs (NMDR) with U-shaped or inverted U-shaped profile.
The case of ETE is well known.
As an example the effects due
to copper deficiency are much
more severe than the ones due
to the its excess.
A dose-response curve is non-monotonic when the slope of the curve
changes sign somewhere within the range of doses examined. Non
monotonicity is not synonymous with low dose, because there are low dose
effects that follow monotonic dose-response curves.
Alternative in vitro methods to characterize the role of EAS
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Non-linearities in the toxicokinetics may be the cause of non monotonic
dose-response relationship NMDR if the MoA is concentration dependent :
two receptors with different actions and different KDs.
two enzymes involved in the biotransformation with different affinity (Km)
producing different metabolites with different effects.
saturation, induction/inhibition of metabolizing enzymes of the unique
metabolic pathway
0.0
1.0
2.0
3.0
4.0
0.0 0.5 1.0 1.5 2.0 2.5
2C19 1A2 2B6
Low doses: the effect due
to metabolites increases
with the dose
High doses: saturation of
metabolites formation-
accumulation of the
parent –possible different
effect or counteracting
effects due to metabolites
Alternative in vitro methods to characterize the role of EAS
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NMDR can be observed in studies where high-doses alters the
experimental model (cell, organ or animal), thus decreasing the observed
response.
This could occur when the formation of aggregates, colloids or micelles at
high concentrations can reduce bioavailability and therefore decrease
the toxicity that appeared at lower concentrations.
The same could happen when cytotoxic doses are tested in in vitro
studies or in vivo, when using doses that are excessively toxic to
animals (doses exceeding the maximum tolerable dose) can reduce the
onset of an effect.
It is likely that these phenomena could contribute to generate
only apparent NMDR.
Alternative in vitro methods to characterize the role of EAS
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The presence of a response at one dose level only is not sufficient to
demonstrate a causal relationship.
A wide dose range and reasonably closely spaced dose intervals (<10-fold
within the same study) is necessary to demonstrate U-shaped dose-
responses.
Poorly described experiments in non-validated models should not be used.
Up to now no scientific consensus has been reached as
to the validity of the studies supporting the ‘low dose
hypothesis ‘
Alternative in vitro methods to characterize the role of EAS
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Extrapolating the windows of exposure in development in animal models to
windows of exposure in human development could be problematic, due to
differences in endocrine signaling across animal species.
Pregnancy:
Circulating estrogen concentrations during pregnancy are ≤100 times lower in
mice than in women pregnant mice may be more susceptible than
pregnant women to the adverse effects of estrogenic compounds
Foetal life:
male rat fetuses are at least an order of magnitude more sensitive than humans
to in utero effects of diethylstilbestrol (DES)
An additional issue is critical windows of exposure,
because of which it may not be possible to identify a
health-based reference value appropriate for the
lifetime of the exposed population.
However, critical time windows are usually
covered by the existing animal testing.
Alternative in vitro methods to characterize the role of EAS
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Difference between endocrine modulation and endocrine disruption.
Many adaptive, compensatory, and physiologically normal/necessary
processes result in measurable endocrine changes.
It is only when these natural mechanisms are affected to such a degree that
adverse effects are induced that ED occurs.
Exogenous chemicals in order to affect the endocrine system must act against
the background of circulating levels of endogenous hormones, which are usually
much more potent than any ED.
The potency issue has to be taken into account in order to understand the
relevance for humans.
Based on estrogenic potency, human exposure to the most potent environmental
estrogens would need to be at least 1000-fold higher than this level, for adverse
effects relevant to the human male to be induced, and such levels of exposure
are remote (Sharpe 2003)
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The most important question to be answered when
the ‘low dose effect’ or NMDR are discussed is :
what is the relevance of low dose effects
observed in animals for the human population?
Biological plausibility must be given and knowing mode of action is a
prerequisite for using the information in risk assessment.
Adversity vs adaptation need to be considered when defining
potential impact on human health
Potency of the exogenous chemical vs endogenous hormones
Statistical plausibility should be also to demonstrate the non-monotonic
nature of each identified dose-response relationship, which is not always an
easy task due to the limited raw data available in the studies published in the
scientific Literature
Levels of human exposure should also be considered in order to determine in
which part of a NMDR exposure occurs.
Alternative in vitro methods to characterize the role of EAS
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To test ED-induced affects postulating a NMDR would imply a change in the
testing strategy: more doses to be tested in order to identify such effects,
especially in the low dose area. To detect small effects at low doses, an
increased number of animals in these dose groups are needed, to strengthen
the statistical power. An optimum of seven doses has been proposed in a recent
EFSA meeting.
Consequences for the RA
This goes in the opposite direction of the EU policy
to reduce animal testing
Alternative in vitro methods to characterize the role of EAS
Studies on kinetics to check internal dose measurements (or cell exposure)
have to be carried out; they would help in producing hypotheses on mode of
action (MoA) and through the use of PBPK modelling in selecting the dose
levels relevant for human exposure.
Proposal for changes in the OECD TG to increase the n° of doses tested while
decreasing the n° of animal/group to derive BMDL rather than NOAEL? Or to
improve study designs incorporating endpoints beyond current OECD methods.
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Questions?
Alternative in vitro methods to characterize the role of EAS
Special thanks to:
the PredictIV team
Emma Di Consiglio
Giuliana Pomponio
To you all for your attention
