INTRODUCTION IN TOXICOLOGY dose reponse relationships

Bangkok Ecotoxicology II 1 INTRODUCTION IN TOXICOLOGY Dose-reponse relationships Bangkok Ecotoxicology II 2 Toxicology: different steps exposure uptake distribution biotransformation interacton with target Effect Fate Toxicokinetics Toxicodynamics dose-response mixtures mode of action Bangkok Ecotoxicology II 3 What do you think is most important to know for risk assessment? The dose (esxposure) makes the poison! Bangkok Ecotoxicology II 4 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 0 150 120 100 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" Bangkok Ecotoxicology II 5 Dose-response curves: two types of response  quantal response  response is yes or no  example: mortality  effect is usually expressed as number of organisms with a response  graded response  response is measured on a gradual scale  example: % inhibition of reproduction, % enzyme inhibition Bangkok Ecotoxicology II 6 Question ?  Do we use quantal response also in human risk assessment? Bangkok Ecotoxicology II 7 How to make a dose-response curve concentration (mg/L) 0 10 20 40 80 160 effect (%) 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) dose 1 1 response Bangkok Ecotoxicology II 8 Quantal dose-response curve: from % to probits % mortality Probits log dosis 1 1 log dosis 100 0 50 5 6 7 4 3 LD50 LD50 sigmoidal curve non-linear regression linear curve linear regression Bangkok Ecotoxicology II 9 Question:  Can you give two good examples of a compounds with a non linear and linear dose response relationship?  Eg. Enzyme induction, mortality for non linear relationship and carcinogenicity for linear relationship Bangkok Ecotoxicology II 10 Some endpoints 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 Bangkok Ecotoxicology II 11 Dose-response curves for quantal response Bangkok Ecotoxicology II 12 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? Bangkok Ecotoxicology II 13 No-observed effect concentration (NOEC) 1. Determine concentration with significant effect (EC) 2. The concentration below the EC is the NOEC 1 1 Effect concentration or dose 10 -8 10 -7 10 -6 10 -5 10 -4 10 -3 10 -2 100 % whole dose-response curve part that is interesting for determining NOEC EC NOEC Bangkok Ecotoxicology II 14 Dose-response curve for graded response: receptor interaction Theoretical background: Receptor theory Receptor R toxic chemical or drug endogenous substance Agonist (A) Effect E Bangkok Ecotoxicology II 15 Receptor theory First step is interaction with a receptor (Receptor is often a protein in cell membrane, nucleus or cytosol) receptor protein active site agonist lock and key concept example recepto: hormone receptor DNA cell membrane a certain protein (enzyme) Bangkok Ecotoxicology II 16 Question: Think about receptors in the organism and come up with two suggestions that are relevant for environmental exposure. Bangkok Ecotoxicology II 17 Different steps in effect: example interaction of hormones Bangkok Ecotoxicology II 18 Different steps in effect: example interaction of hormones Bangkok Ecotoxicology II 19 Bangkok Ecotoxicology II 20 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 Globulin e.g. SHBG / ABP α -fetoprotein CBP TBP crosstalk e.g. between: - membrane receptors - nuclear receptors - Ah receptor ligand binding gene expression cellular effects e.g. cell proliferation cell differentiation gamete maturation tissue effects endogenous proteins e.g. vitellogenin progesterone receptor recombinant reporter genes mammalian cells yeast Endocrine Chemical Steroidogenesis Receptors e.g. oestrogen receptor androgen receptor e.g. aromatase metabolic enzymes e.g. P450s membrane e.g. SHBG membrane receptor R A AR [...]... of affinity of a compound (A) to the receptor (in vitro) Binding of compounds to the receptor can be measured in vitro Ka A+R k AR Effect Differences in binding affinities are expressed in Ka (affinity constant) Ka = [AR] / ( [A] [R]) Kd (dissociation constant) = 1 / Ka Relation between the Effect (E) and dose ([A]) can be expressed as: E/Emax = [A] / (Kd + [A]) Bangkok Ecotoxicology II 22 Dose- response... antagonists Bangkok Ecotoxicology II 30 Actual activity or effect concentration depends on: 1 Intrinsic activity active site in vitro agonist receptor protein 2 Concentration at target uptake distribution biotransformation in vivo Bangkok Ecotoxicology II 31 Question? What type of in vitro experiment could you use to study: Metabolism in the liver Binding to an estrogen receptor Bangkok Ecotoxicology II 32... Bangkok Ecotoxicology II 25 Binding affinities of estradiol, DES and hydroxylated biphenyls Steven P Bradbury et al Environmental Toxicology and Chemistry: Vol 15, pp 1945–1954 Bangkok Ecotoxicology II 26 Agonist versus antagonist Agonist: interacts with the receptor (active site) resulting in an effect Antagonist: interacts with the receptor with no (or other) effect: interaction of an antagonist... and inhibitor of MFO system conc of parathion MFO system parathion > paraoxon (inhibits acetylcholine-esterase) inhibitor of oxidative enzymes: salicylamide Bangkok Ecotoxicology II 38 Example of synergism parathion parathion and inducer of MFO system effect conc of parathion MFO system parathion > paraoxon (inhibits acetylcholine-esterase) inducer of oxidative enzymes: PCBs Bangkok Ecotoxicology... protein binding in blood) Bangkok Ecotoxicology II 36 Biotransformation of organophosphates Synergism and antagonism often via influence on biotransformation: Example: biotransformation of organophosphates OCH 3 P=S OCH 3 O OCH 3 oxidation (NADPH) O P=O OCH 3 oxon metabolite de-alkylation GSH specific mode of action: inhibition of Acetylcholin-esterase by oxon metabolite OCH 3 O P=S OH Bangkok Ecotoxicology... Bangkok Ecotoxicology II 23 Question Think about a group of compounds that work through the same receptor but in which individual compounds have different potencies Dioxins and PCBs on the Ah-receptor or organophosphate esters with acetylcholinesterase Bangkok Ecotoxicology II 24 Structures of estradiol, DES and polychlorinated biphenyls 1 estradiol 2 DES 3-14: hydroxylated PCB’s Bangkok Ecotoxicology... agonist receptor protein Bangkok Ecotoxicology II 27 Competitive antagonism (reversible) active site shift in dose- response curve antagonist agonist receptor protein A 100 % A with Antagonist Effect or [AR] 1 1 1 1 10-8 10-7 10-6 10-5 10-4 10-3 Bangkok concentration (mol/L) Ecotoxicology II 28 Non-competitive antagonism (irreversible) active site antagonist shift and decrease in dose- response curve agonist... A 1.0 TU Bangkok Ecotoxicology II 35 Synergism / antagonism In general: 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... decrease in dose- response curve agonist receptor protein A 100 % A with Antagonist Effect or [AR] 1 1 1 1 10-8 10-7 10-6 10-5 10-4 10-3 Bangkok Ecotoxicology II concentration (mol/L) 29 Importance of agonists or antagonists In pharmacology: agonists: for example estrogens as contraceptives, drugs in certain cancers antagonists: for example at hyperactivity In toxicology: Many chemicals have a receptor mediated... 0.5 1.0 Sum of toxic units: measure for joint toxicity Bangkok Ecotoxicology II 33 Simple similar action: concentration additivity Mixture of A and B: A concentration (in TU) B mixture 0.12 0.25 0.5 1.0 0.12 0.25 0.5 1.0 0.24 0.5 1.0 2.0 % effect 0 10 50 100 LC50 of mixture Effect of mixtures at sum TU = : concentration-addition Bangkok Ecotoxicology II 34 Joint effects (same response) of mixtures of . Bangkok Ecotoxicology II 1 INTRODUCTION IN TOXICOLOGY Dose- reponse relationships Bangkok Ecotoxicology II 2 Toxicology: different steps exposure uptake distribution biotransformation interacton. Ecotoxicology II 22 Measurement of affinity of a compound (A) to the receptor (in vitro) A + R AR Effect Binding of compounds to the receptor can be measured in vitro  Differences in binding. 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) dose 1 1 response Bangkok Ecotoxicology