HLTH 340 Lecture A2 Toxicokinetic processes: absorption (part-1) NOTICE: These materials are subject to Canadian copyright and are presented here as images published in journals and books for which the University of Waterloo holds a licensed electronic subscription. These materials are provided to HLTH 340 students for their exclusive use though a non-public courseware system (UW-LEARN) and the images are restricted to the use of HLTH 340 students. Reproduction, transmittal, copying, or posting of these images by students in any form, electronic or physical, is strictly prohibited.

Basic Steps in Toxicological Analysis

Toxicokinetic processes- also termed pharmacokinetics, ADME, disposition toxicokinetics describes the movement of xenobiotic substances into and within the organism subsequent to an environmental exposure descriptive (semi-quantitative) analysis quantitative analysis (mathematical formulas and graphs) computer-based simulations (PB-PK models = physiologically-based pharmacokinetic models) Absorption controls entry of xenobiotics through the external membrane barriers into the blood (or lymphatic) circulation local effect (tissues near site of absorption) regional effect (tissues downstream from site of absorption -- “first-pass effects” systemic effect (throughout the body) Distribution determines the movement of xenobiotic molecules with the circuatory fluids and specific organs and tissues Metabolism (biotransformation) describes the biochemical processes that convert the original (parent) xenobiotic to various metabolic products (metabolites) Excretion controls the removal of the xenobiotic or its metabolites from the body

Toxicokinetic (ADME) processes

Toxicokinetic and toxicodynamic pathways jointly affect toxicity

Route of exposure Route of exposure The ROUTE (site) of exposure is an important determinant of the ultimate DOSE – different routes may result in different rates of absorption. Dermal (skin) Inhalation (lung) Oral (GI) Injection The ROUTE of exposure may be important if there are tissue-specific toxic responses. Toxic effects may be local (in a specific tissue) or systemic (throughout the organism)

Routes of Absorption, Distribution and Excretion first-pass effect distribution excretion

First-pass extraction: the hepatic portal vein carries absorbed nutrients and xenobiotics to the liver

Absorption of molecules across external and internal membrane barriers passive diffusion (non-selective) receptor-mediated transport (selective) transcellular paracellular

Types of membrane transport mechanisms: active transport and passive transport external dose (site of absorption) internal dose (blood) external dose (site of absorption)

intercellular tight junction (can be open, closed, or ‘leaky’ Intestinal absorption via passive diffusion using paracellular and transcellular permeation pathways intercellular tight junction (can be open, closed, or ‘leaky’

Paracellular permeation through a membrane barrier occurs between adjacent cell membranes apical (outside) The characteristics of the paracellular pathway are defined by specific junctional complexes that span the intercellular space. There are four types of complexes: zona occludens, or tight junction; zona adherens, or intermediate junction; desmosomes; and gap junctions. Specific proteins localized to each complex link adjacent cells and the cytoskeleton. Original models of the paracellular pathway as a static barrier are being replaced by a more dynamic model in which the junctional complexes are involved in signaling and regulation, most likely through protein phosphorylation or dephosphorylation. The tight junction is the most apical complex and is believed to control permeability across the paracellular pathway through a series of strands and grooves. Molecular definition of the specific components of the tight junction ( eg , Z0-1, Z0-2, occludin, cingulin) may permit a clearer understanding of how the tight junction functions as a barrier for ions and macromolecules. baso-lateral (inside)

The tight junction (TJ) barrier structure forms pore structures between adjacent cell membranes The TJ barrier consists of two components — physiological pores and pathological breaks. All epithelial TJs have a system of small approximately 8-angstrom pores that varies among cell types in ionic charge selectivity and in porosity, i.e. the apparent number of pores. The mechanism controlling overall porosity is unclear, but it is known that preferences for ionic charges is controlled by claudins. The claudins form the pore structure or influence their size and shape. Each claudin has a characteristic influence on the permeability for small cations and anions. The passage of material larger than approximately 8-angstroms shows no charge selectivity. This small pathway may represent a pathological break between cells. Such disruptions can arise in response to proinflammatory factors like interferon-gamma and tumor necrosis factor-alpha. claudins

Transcellular passive diffusion is the commonest type of absorption across membrane barriers passive diffusion - a process that requires no molecular transport system or energy source (random migration by individual solute molecules) passive diffusion cannot concentrate substances across membrane barrier (no pumping action) bidirectional -- flow of molecules will follow the concentration gradient in either direction (in or out of tissue) absorption rate for passive diffusion is determined by 3 major factors surface area through which diffusion is occurring (membrane lining of gut, lung, and skin) concentration gradient [Cexternal] >> [Cinternal] permeability of the substance through the membrane barrier permeability is typically determined by each substance’s physicochemical properties molecular weight • smaller molecules (MW < 500 daltons) are often able to migrate through biomembranes by passive diffusion • over 80% of effective drugs have a MW < 450 daltons hydrophobicity tendency of a substance to dissolve preferentially in fatty or oily biological media, but not in water ionization • molecules that carry positively or negatively charged functional groups have ionic properties • charged ionic groups experience electrostatic interactions with ionic phospholipid membrane groups polarity (hydrogen bonding) molecules with uneven electrical charge distribution (polar compounds) form H-bonds with water

Lipid sieve model of cell membrane The ‘lipid sieve’ model helps to explain how small molecules that are lipophilic can permeate through the cellular phospholipid membrane by passive diffusion hydrophilic molecules cannot permeate the membrane unless there is a specific paracellular transport channel or membrane-associated active transport pump.

Molecular dynamics computer simulation of membrane diffusion during xenobiotic absorption

Lipophilic and hydrophilic solubility lipid solubility affects transcellular passive diffusion through the phospholipid biomembranes hydrophilic (water soluble) ionic molecules carry one or more positive or negative charges polar molecules carry partial positive or negative charges phospholipid molecules on the membrane surface contain a zwitterionic charge distribution negatively charged phosphate groups PO4-- positively charged choline groups N-[CH3]4+ charged phospholipid groups will repel or bind ionic hydrophiles via electrostatic interactions charged phospholipid groups will form hydrogen bonds (H-bonds) with uncharged hydrophiles that have polar functional groups (esters, amides, etc.) most hydrophiles cannot pass across membranes by transcelluar passive diffusion lipophilic (fat and oil soluble) electrically neutral molecules with no positive or negative charges no electrostatic repulsion or H-bond attraction at the membrane surface readily penetrate into and through the the non-polar interior of biomembranes many small lipophiles can pass through biomembranes by transcellular passive diffusion usually small lipophiles can be more readily absorbed than most small hydrophiles lipophilicity factors are used to predict passive absorption of drugs and xenobiotics lipophilicity = hydrophobicity - [polarityH-bonding + ionic interactions] calculated rate of absorption = 1/size (MW) x 1/lipophilicity (log Ko/w)

Partition coefficient is a quantitative measure of the degree of lipophilicity of a given molecule partition coefficient (Kp, Ko/w) measures relative degree of solubility in lipid (lipophilicity) and water (hydrophilicity) measure concentration of xenobiotic in 2-phase solvent mixture oily non-aqueous phase solvent (octanol) and watery aqueous phase (H2O) ‘oil and water don’t mix’ Ko/w = conc (octanol) / conc (water) Ko/w > 1 is lipophilic Ko/w<1 is hydrophilic Ko/w = 0 - 1 is amphiphilic (mixed) log Ko/w often expressed in log10 units example: Ko/w = 1000 --> log Ko/w = 3 (strongly lipophilic)

Lipinski’s ‘rule of five’ for predicting xenobiotic absorption by transcellular passive diffusion Poor transcellular absorption and membrane permeation is more likely when: there are more than 5 H-bond donors in the molecular structure (mainly OH and NH groups) the molecular weight is over 500 the molecule’s log Ko/w is over 5 there are more than 10 H-bond acceptors in the molecular structure (mainly N and O containing polar groups)

Effect of lipophilicity on the absorption rate of 3 related xenobiotic substances (barbiturate drugs) ko/w ko/w ko/w

Effect of partition coefficient on absorption rate extremely lipophilic strongly lipophilic 4 - 5 3 2 moderately lipophilic log Kp > 5 substances are poorly absorbed due to membrane trapping or lack of water solubility 1 log Kp < 0 substances are poorly absorbed due to ionic interactions or H-bonding 0 - 0.9 mixed or amphiphilic hydrophilic < 0

Absorption of large or non-permeable xenobiotic molecules can occur via cellular endocytosis

Absorption into brain of manganese (Mn2+) ions via active transport channels and cellular endocytosis TMI slide (illustrative purposes only)