INTRODUCTION IN TOXICOLOGY dose reponse relationships

Bangkok Ecotoxicology II 7How to make a dose-response curve concentration mg/L 0 10 20 40 80 160effect % 0 0 5 45 90 100 Dose-response curve: relation between effect and dose at a certai

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INTRODUCTION IN TOXICOLOGY Dose-reponse relationships

Toxicology: different steps

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What do you think is most important to know for risk

assessment?

The dose (esxposure) makes the poison!

Example of influence dose on response

coma

alcohol consumption (g) plasma peak concentration

3.5 3.0 2.5 2.0 1.5 1.0 0.5

150 120 100

60

30 20 10

intoxication

decreased reaction speed

Paracelsus (16e eeuw):

"Alle Dinge sind Gift allein die Dosis macht das ein Ding kein Gift ist"

ƒ response is measured on a gradual scale

ƒ example: % inhibition of reproduction, % enzyme inhibition

Question ?

‹ Do we use quantal response also in human risk assessment?

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How to make a dose-response curve

concentration (mg/L) 0 10 20 40 80 160effect (%) 0 0 5 45 90 100

Dose-response curve:

relation between effect and dose

at a certain exposure duration

system:

in vivo (whole organisms).

or in vitro (cells, protein)

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sigmoidal curve

non-linear regression

linear curvelinear regression

LOED: lowest effective dose

LOEC: lowest effective concentration

ED50: dose with 50 % effect

EC50: concentration with 50 % effect

LD50: dose that causes 50 % mortality

LC50: concentration that causes 50 % mortality

NOEC: No observed effect concentration

NOEL: No observed effect level

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Dose-response curves for quantal response

Questions:

‹ Determine in the dose response curves the LOEC and LOED

‹ Why is the EC100 not similar to the LOED in the dose

responses?

‹ What would be the EC75 and ED75 in a dose response

relationship?

‹ If a benchmark dose would be an EC0.5, what would this mean?

‹ Is the observed LOED or LOED similar to the real lowest

effective dose or concentration?

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No-observed effect concentration (NOEC)

1 Determine concentration with significant effect (EC)

2 The concentration below the EC is the NOEC

whole dose-response curve

part that is interesting for determining NOEC

ECNOEC

Dose-response curve for graded response: receptor

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Receptor theory

First step is interaction with a receptor (Receptor is often a protein

in cell membrane, nucleus or cytosol)

cell membrane

a certain protein (enzyme)

Question:

Think about receptors in the organism and

come up with two suggestions that are

relevant for environmental exposure.

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Different steps in effect: example interaction of hormones

Different steps in effect: example interaction of hormones

Different steps in effect: example interaction of hormones

Potential targets and mechanisms of action

of endocrine modulators Endocrine modulators may elicit adverse affects through a number of different mechanisms such as interactions with binding globulins, inhibition of steroidogenic enzymes and/or binding to receptors Crosstalk occurs when mechanism intersect to elicit unique responses that may modulate endogenous gene expression The figure illustrates that a

number of in vitro assays can be developed

to identify and assess endocrine modulators provided that the mechanism of action is

known However, it is unclear if in vitro assays can accurately predict in vivo

responses due to their lack of metabolic

activity and therefore, in vitro data requires verification in vivo.

Binding

CBP TBP

crosstalk e.g between:

R

A

AR

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Dose-response curve for graded response:

receptor theory

Assumptions:

– Interaction between A and R is reversible

– Effect is related to concentration of occupied receptors: [AR]

– Effect is maximal when all receptors are occupied: Emax= a.[R]t, where [R]t is the total concentration of receptors

Measurement of affinity of a compound (A) to the

receptor (in vitro)

Binding of compounds to the receptor can be measured in vitro

ƒDifferences in binding affinities are expressed in Ka (affinity constant)

‹ Dioxins and PCBs on the Ah-receptor or organophosphate

esters with acetylcholinesterase

Binding affinities of estradiol, DES and hydroxylated

biphenyls

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Agonist versus antagonist

Agonist: interacts with the receptor (active site) resulting in an effectAntagonist: interacts with the receptor with no (or other) effect:

interaction of an antagonist may be

1 competitive (reversible) or,

Competitive antagonism (reversible)

receptor

protein

active site

agonistantagonist

Effect or[AR]

100 %

Antagonistshift in

dose-response curve

10-8 10-7 10-6 10-5 10-4 10-3

100 %

A

A with Antagonist

concentration (mol/L)

1 1

Importance of agonists or antagonists

In pharmacology:

agonists: for example estrogens as contraceptives, drugs in certain cancersantagonists: for example at hyperactivity

In toxicology:

Many chemicals have a receptor mediated toxicity

Chemicals may affect processes as agonists or antagonists

‹ What type of in vitro experiment could you use to study:

‹ Metabolism in the liver

‹ Binding to an estrogen receptor

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Evaluation of effects of mixtures: toxic units (TU)

Toxic units: concentrations of chemicals are expressed as fractions of their effect concentrations

Sum of toxic units: measure for joint toxicity

Simple similar action: concentration additivity

‹ one chemical A with a certain response

‹ other chemical B enhances or inhibits the activity of chemical A, without having the response itself

Examples:

‹ at level of biotransformation: chemical A has to be activated to exert toxicity / chemical B induces activity of enzyme / results in potentiation

‹ at level of interaction with a receptor

‹ at level of bioavailability (e.g protein binding in blood)

specific mode of action:

parathion -> paraoxon (inhibits acetylcholine-esterase)

MFO system

parathion -> paraoxon (inhibits acetylcholine-esterase)

inducer of oxidative enzymes: PCBs

acetaldehyde dehydrogenase

ADH

Dithiocarbamates inhibit ADH

aldehydes are strong irritating chemicals

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Question?

‹ Could you give an example of a practical situation in which

antagonism is used?