Chemical pollutants of the food chain.

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

2. Type of contaminations ENVIRONNEMENT (water/soil/air) Végétal Animal 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. 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) Plasma transportation In the blood the molecule can be free or bound Binding can occur with specific or non specific transporters

6. Modulation of toxicity (3) 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

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

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

12. The relevance of animal model: analysis of the case of the evaluation of fipronil as a thyroid disruptor With is the pathophysiological scheme of action of fipronil as a thyroid disruptor considered as non relevant to human? Fipronil PL T3, T4 free T3, T4 bound Clearance • TBG expression: • protects TH from peripheral elimination • Pool of TH Bound T4: TBG 73% 53% 0% in adult TTR 19% 36% 85% Albumine 8% 11% 15%

13. The sheep as a good model to study thyroid disruptors? OR? Not to be 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 ?

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 (%) 0.02 0.04 0.07 • T4 half-life (Days) 2 5-9 0.5 • TBG • T4 Dissociation constant (nM) 0.112 0.105 NA/adult • T4 Maximal binding capacity(nm/l) 160 266 • TTR • T4 Dissociation constant (nM) 7.14 6.25 2.78 • T4 Maximal binding capacity(nm/l) 4494 3230 3968

15. THX+ T3 * Vehicle 0.8 0.04 240 80 Fipronil 0.6 0.03 60 160 Total T4 (ng/ml) 0.4 0.02 40 Total T4 (ng/ml) (mL/min/kg) (mL/min/kg) 80 0.01 0.2 20 0 0 0.00 0 0 10 20 30 Vehicle Fipronil before after 0 25 50 75 100 125 Temps (h) 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 10000 Fipronil administrations Sulfone Fipronil sulfone 1000 1000 Plasma concentrations (ng/mL) 100 100 Fipronil Fipronil 10 10 0 5 10 15 20 0 5 10 15 20 25 30 35 Temps (j) Time (days)

17. 10000 10000 1000 (ng/ml) Plasma concentration 1000 (ng/ml) Plasma concentrations 100 100 Fipronil Sulfone Fipronil Sulfone 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 Sulfone/FIP= 4 Sulfone/FIP>100 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)

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.