Toxicokinetics Toxicodynamics

1. Toxicokinetics & Toxicodynamics Toxicokinetics (Determines the no. molecules that can reach the receptors) Uptake Transport Metabolism & transformation Sequestration Excretion Toxicodynamics (Determines the no. of receptors that can interact with toxicants) Binding Interaction Induction of toxic effects

2. Uptake and Elimination  

3. Toxicokinetics Uptake Transport Metabolism & Transformation Sequestration Excretion

4. Uptake routes Ingestion (toxicity may be modified by enzymes, pH and microbes) Respiration (Air borne toxicants) Body surface (Lipid soluble toxicants such as carbon terta chloride and organophosphate)

5. Uptake Barriers Cell membrane Cell wall/cuticles/stomata Epithelial cells of GI tract Respiratory surface (lung, gill tracheae) Body surface

7. Uptake of Toxicants Passive diffusion Facilitated transport Active transport Pinocytosis

8. Uptake by Passive diffusion Uncharged molecules may diffuse along conc. gradient until equilibrium is reached Not substrate specific Small molecules of < 0.4 nm (e.g. CO, N20, HCN) can move through cell pores Lipophilic chemicals may diffuse through the lipid bilayer

10. Uptake by Passive diffusion First order rate process, depends on: Concentration gradient Surface area (aveoli = 25 x body surface) Thickness (fluid mosaic phospholipid bi-layer ca. 7 nm) Lipid solubility & ionization(dissolved before transport, polar chemicals have limited diffusion rate) Molecular size (membrane pore size = 4-40 A, allowing MW of 100-70,000 to pass through)

11. Diffusion governed by Flicks law   D/dt = KA (Co - Ci) / X  Where: dD/dt = rate of transport accross the membrane K= constant A= Cross sectional area of membrane exposed to the compound Co = Concentration of the toxicant outside the membrane Ci = Concentration of the toxicant inside the membrane X= Thickness of the membrane

12. Uptake by Facilitated Transport Carried by trans-membrane carrier along concentration gradient Energy independent May enhance transport up to 50,000 folds Example: Calmodulin for facilitated transport of Ca

13. Uptake by Active Transport Independent of or against conc. gradient Require energy Substrate –specific Rate limited by no. of carriers Example: P-glycoprotein pump for xenobiotics (e.g. OC) Ca-pump (Ca2+ -ATPase)

15. Uptake by Pinocytosis For large molecules ( ca 1 um) Outside: Infolding of cell membrane Inside: release of molecules Example: Airborne toxicants across alveoli cells Carrageenan accross intestine

17. Transport & Deposition Transport Blood Lymph, haemolymph Water stream in xylem Cytoplamic strands in phloem Deposition Toxicant Target organs Pb Bone, teeth, brain Cd Kidney, bone, gonad OC, PCB Adipose tissue,milk OP Nervous tissue Aflatoxin Liver

18. Metabolism & Transformation Evolved to deal with metabolites and naturally occurring toxicants Principle of detoxification: Convert toxicants into more water soluble form (more polar & hydrophilic) Dissolve in aqueous/gas phases and eliminate by excretion (urine/sweat) of exhalation Sequestrate in inactive tissues (e.g bone, fat)

19. P450 system A heme-containing cytochrome protein located in ER, and is involved in electron transport. Highly conservative, occur in most plants & animals Two phases of transformation May increase or decrease toxicity of toxicants after transformation (e.g turn Benzo[a]pyrene into benzo[a]pyrene diol epoxide, and nitroamines into methyl radicals) Inducible by toxicants

20. Induction of P450

21. Phase I Transformation Mixed Function Oxidase (MFO) System in smooth ER is responsible (Microsomes) In vertebrates, primarily found in liver parenchyma cells, but also other tissues (e.g intestine, gill) In invertebrates, found in hepatopancrease & digestive glands Lower MFO activities in molluscs Add polar group(s) to increase hydrophilicity for Phase II transformation

22. Examples of Phase I Transformation Hydrolysis   RCOO-R’ + H2O ---------> RCOO-H + R’-OH Hydroxylation   NADP NADP+ R-H --------------------------> R-OH + H2O

23. Examples of Phase I Transformation Epoxidation O R-CH==CH-R’ -----------> R---CH ----CH-R’  

24. Phase II transformation Cytochrome P450 II enzyme systems in cytosol is responsible Covalent conjugation to water soluble endogenous metabloites (e.g. sugars, peptides, glucuronic acid, glutathione, phosphates & sulphate) May involve deamination, acyclic hydroxylation, aromatic hydroxylation, and dealkylation Further increase hydrophilicity for excretion in bile, urine and sweat

25. Important Phase II enzymes Glutathion S-transferases (GST) Epoxide Hydrolase (EH) UDP-glucuronosyltransferase (UDP-GTS) Sulfotransferase (ST).

26. Examples of Phase II Transformation Deamination   R-NH2 ---------------------------> R=O + NH3  

27. Examples of Phase II Transformation Dealkylation R-CH2-CH3 ----------------------> R + CH3-CH2O Dehalogenation:   R-Cl ---------------------------------> R-H + Cl+

28. Glutathione-S-transferase (GST)   O R------R’ ----------------------> HO-R-SG   R-Cl ------------------------------> R-SG + Cl

29. Sequestration Animals may store toxicants in inert tissues (e.g. bone, fat, hair, nail) to reduce toxicity Plants may store toxicants in bark, leaves, vacuoles for shedding later on Lipophilic toxicants (e.g. DDT, PCBs) may be stored in milk at high conc and pass to the young Metallothionein (MT) or phytochelatin may be used to bind metals

30. Excretion Gas (e.g. ammonia) and volatile (e.g. alcohol) toxicants may be excreted from the gill or lung by simple diffusion Water soluble toxicants (molecular wt. < 70,000) may be excreted through the kidney by active or passive transport Conjugates with high molecular wt. (>300) may be excreted into bile through active transport Lipid soluble and non-ionised toxicants may be reabsorbed (systematic toxicity)

31. Tutorial Questions Find TWO enzymes/proteins which are inducible by xenobiotics or metals Molluscs have low P450 activities. They are often used as pollution indicators for metals and xenobiotics. Explain why. Lipophilic compounds may normally have a longer biological half life. Explain why. Why exposure of animals to sub-lethal level of toxicants may increase tolerance of the organisms to the chemical.