1. Chemical pollutants of the food chain. Catherine Viguié CR INRA

2. Type of contaminations ENVIRONNEMENT (water/soil/air) VégétalAnimal Human

3. Pollutants evolution in the environnement Different pathways for molecule chemical transformations Abiotic (oxidation – light-unduced) Biotic (through alive organism from bacterias to and vegetal organisms) Consequences: From one initial molecule to numerous metabolites Inactivation (liver metabolism) Bioactivation (metaboliste more toxic than the first molecule)

4. Modulation of toxicity (1) Transport mechanisms through the Physiological barriers Passive diffusion Active transporters (specifics) Efflux pumps Physical barriers (tight junctions) ABSORPTION (Digestive tract) Determining step for blood concentrations: global exposure Exposure of target tissues Brain Placenta-Foetus Potential for toxicity Competition for transporters

5. Plasma transportation In the blood the molecule can be free or bound Binding can occur with specific or non specific transporters Limiting factor for clearance mechanisms highly bound molecules to specific transporters (binding proteins) : high potential for bioaccumulation Potential Toxicity Competition with specific binding protein of endogenous molecule such as hormones will be associated to an increase in hormone clearance Modulation of toxicity (2)

6. METABOLISM Phase I (cyt P450): enzymes Phase II Elimination (kidney – liver) BIOACTIVATION vs. DETOXIFICATION Limiting factor for the elimination of the xenobiotic Bioaccumulation Potential toxicity Competition/inhibition of enzymes Induction of enzymes Modulation of toxicity (3)

7. Mechanisms and sites of action Endocrine disruptors Metabolism of hormones Transportation Receptors Hormone synthesis Pathogens (bacterias- parasites) Resistance to therapeutic agents Central nervous systems Neurodegenerative diseases Alteration of the development of the central nervous system Cancers

8. Effects are dose and time dependent Oral contamination very low doses + Long period exposure Mecanisms of action Critical period

9. The relevance of animal model for the risk analysis of food contaminant for human health Transport mechanisms through the Physiological barriers  placenta  efflux pumps Metabolism of the toxic Physiology of the altered function:  Plasma binding  Neuroregulation  Hormone metabolism = All these phenomenon = causes for interspecies differences in the sensitivity to toxic effects of xenobiotics => Need for relevant model for human from the standpoints of:  the metabolism of the xenobiotic  the regulatory scheme of the function

10. The thyroid function Hypothalamus TRH Thyroïd TPO TG NIS Pituitary TSH bound TH (T3 T4) blood Clearance - free T3, T4 HOT spot 1 HOT spot 2 Whole body / all life effects HOT spot 3 Hot spots: debates on the relevance of animal models 80% of circulating T3

11. The relevance of animal model: analysis of the case of the evaluation of fipronil as a thyroid disruptor TRH TSH Thyroid Hypothalamus Anterior pituitary T3, T4 free T3, T4 bound PL Clearance - Fipronil Increased T4 clearance // hepatic enzyme induction Fipronil and thyroid disruption in the rat 0 0.2 0.4 0.6 0.8 SolvantFipronil (mL/min/kg) *

12. The relevance of animal model: analysis of the case of the evaluation of fipronil as a thyroid disruptor T3, T4 free T3, T4 bound PL Clearance Fipronil With is the pathophysiological scheme of action of fipronil as a thyroid disruptor considered as non relevant to human? TBG expression: protects TH from peripheral elimination Pool of TH Bound T4: TBG73%53%0% in adult TTR19%36%85% Albumine8%11%15%

13. To be OR ? Not to be The question is : What is the relevance of animal models for an endocrine system that exhibits multiple interspecies particularities in its regulatory scheme ? The sheep as a good model to study thyroid disruptors?

14. The relevance of animal model: analysis of the case of the evaluation of fipronil as a thyroid disruptor The protective role of specific thyroid hormone binding protein TBG Free T4 fraction (%)0.020.040.07 T4 half-life (Days)25-90.5 TBG T4 Dissociation constant (nM)0.1120.105NA/adult T4 Maximal binding capacity(nm/l) 160266 TTR T4 Dissociation constant (nM)7.146.252.78 T4 Maximal binding capacity(nm/l)449432303968

15. The relevance of animal model: analysis of the case of the evaluation of fipronil as a thyroid disruptor Does the effect of fipronil on thyroid function differ between rat en sheep accordingly to the assumes protective role of TBG? YES

16. The relevance of animal model: analysis of the case of the evaluation of fipronil as a thyroid disruptor Is the interspecies difference in TBG expression the only explanation for the diffrential effect of fipronil on thyroid function between rat and sheep? The role of fipronil metabolic pathways 10 100 1000 10000 05101520 Time (days) Plasma concentrations (ng/mL) Fipronil administrations Fipronil Fipronil sulfone 10 100 1000 05101520253035 Temps (j) Sulfone Fipronil

17. The relevance of animal model: analysis of the case of the evaluation of fipronil as a thyroid disruptor Is the interspecies difference in TBG expression the only explanation for the diffrential effect of fipronil on thyroid function between rat and sheep? The role of fipronil metabolic pathways Hypothesis: transformation of fipronil in fipronil sulfone (hapatic cytochromes) = bioactivation relative to potential thyroid toxicity. Sensitivity to fipronil as a thyroid disruptor is modulated by hepatic metabolism of fipronil (‡ between species) 100 1000 10000 FipronilSulfone Plasma concentration (ng/ml) 100 1000 10000 FipronilSulfone Plasma concentrations (ng/ml) Sulfone/FIP= 4 Sulfone/FIP>100

18. Conclusion Never forget the physiology (function & metabolism) The relevance of experimental animal model should always be addressed carefully Necessity to develop and adapt these models to allow long term low dose exposure studies relevant to human exposure The need for physiologically-based models allowing a global assessment of endocrine function with a predictive value and mechanistical outlets.