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 K1 Biological System K2 Elimination Uptake K1 > K2 : Accumulation & Toxic effect

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 Toxicant Aryl Hydrocarbon Receptor Toxicant-Receptor Complex Bind at Specific site hours Translocating protein m-RNA for CYP1A

21. Phase I Transformation • Mixed Function Oxidase (MFO) System insmooth 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 • EpoxidationO 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 GST GST

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.