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

Uptake and Elimination

Uptake
K1

Biological System

K2

Elimination

K1 > K2 : Accumulation & Toxic effect

Toxicokinetics
1. 2. 3. 4. 5. Uptake Transport Metabolism & Transformation Sequestration Excretion

Uptake routes
1. Ingestion (toxicity may be modified by enzymes, pH and microbes) 2. Respiration (Air borne toxicants) 3. Body surface (Lipid soluble toxicants such as carbon terta chloride and organophosphate)

Uptake Barriers
1. Cell membrane
2. Cell wall/cuticles/stomata

3. Epithelial cells of GI tract

4. Respiratory surface (lung, gill

tracheae) 5. Body surface

Uptake of Toxicants

1. 2. 3. 4.

Passive diffusion Facilitated transport Active transport Pinocytosis

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

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)

Diffusion governed by Flicks law

Where:

D/dt = KA (Co - Ci) / X

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

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

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)

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

Transport & Deposition

Transport
Blood Lymph, haemolymph Water stream in xylem Cytoplamic strands in phloem

Deposition
Toxicant Pb Cd OC, PCB OP Aflatoxin Target organs Bone, teeth, brain Kidney, bone, gonad Adipose tissue,milk Nervous tissue Liver

Metabolism & Transformation

Evolved to deal with metabolites and naturally occurring toxicants Principle of detoxification:
1. Convert toxicants into more water

soluble form (more polar & hydrophilic) 2. Dissolve in aqueous/gas phases and eliminate by excretion (urine/sweat) of exhalation 3. Sequestrate in inactive tissues (e.g bone, fat)

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

Induction of P450
Toxicant Aryl Hydrocarbon Receptor

Toxicant-Receptor Complex

Bind at Specific site

hours

Translocating protein m-RNA for CYP1A

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

Examples of Phase I Transformation

Hydrolysis
RCOO-R + H2O ---------> RCOO-H + R-OH

Hydroxylation
NADP NADP+

R-H --------------------------> R-OH + H2O

Examples of Phase I Transformation

Epoxidation
O R-CH==CH-R -----------> R---CH ----CH-R

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

Important Phase II enzymes

Glutathion S-transferases (GST) Epoxide Hydrolase (EH) UDP-glucuronosyltransferase (UDPGTS) Sulfotransferase (ST).

Examples of Phase II Transformation

Deamination
R-NH2 ---------------------------> R=O + NH3

Examples of Phase II Transformation

Dealkylation R-CH2-CH3 ----------------------> R + CH3-CH2O

Dehalogenation:
R-Cl ---------------------------------> R-H + Cl+

Glutathione-S-transferase (GST)
O GST R------R ----------------------> HO-R-SG

GST

R-Cl ------------------------------> R-SG + Cl

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

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)

Tutorial Questions
1. Find TWO enzymes/proteins which are inducible by xenobiotics or metals 2. Molluscs have low P450 activities. They are often used as pollution indicators for metals and xenobiotics. Explain why. 3. Lipophilic compounds may normally have a longer biological half life. Explain why. 4. Why exposure of animals to sub-lethal level of toxicants may increase tolerance of the organisms to the chemical.