AH 3175E Module 6

substrate (trailer): glutathione (GSH)

substrate (Phase II Enzymes/substrate/donor

acetyltransferasetrailer): glutathione (GSH)

substrate donor (cofactor): itself

substrate donor (cofactor): itself

elements

pure chemical substances with specific substances, defined by the number of protons

atoms

the smallest units of elements that still retain the element's properties

atoms contain electrons, neutrons, and protons

molecule

a group of two or more atoms held together by chemical bonds

molecules can be made of the same element or multiple elements

compound

a molecule that contains multiple elements

99% of the body is made of

carbon, hydrogen, nitrogen, oxygen, calcium, phosphorus, sulfur

size and composition of compounds in the body

varies drastically

proteins are made up of

amino acids

average protein

46 kg/mole

average amino acid

110 g/mole

structure of molecules determined

toxicity

what is pKa

pKa = -log10(Ka)

measure of acidity of a molecule

convenient way to identify if a molecule will be protonated or deprotonated in a solution of a specific pH

the lower the pKa

the stronger the acid

Ka

acid dissociation constant

pKa < pH

pKa > pH

pKa = pH

pKa < pH: most molecules in solution will be deprotonated

pKa > pH: most molecules in solution will be protonated

pKa = pH: half of molecules in solution will be protonated, half will be deprotonated

why is the pKa of different molecules important?

different body fluids have different pH values

charge on a molecule impacts its transport

n-octanol/water partitioning

determines if a compound is hydrophilic or lipophilic

n-octanol/water partitioning is determined by

1. add chemical of unknown logP

2. mix thoroughly

3. remove a sample from each layer

4. measure the concentration of the chemical in both octanol and water

5. calculate ratio of the chemical in octanol versus water

lipophilic compounds

preferentially dissolve in n-octanol over water

tend to be non-polar compounds such as hydrocarbon-based molecules

hydrophilic compounds

preferentially dissolve in water over n-octanol

tend to be polar compounds, such as glucose or NaCl

partition coefficient

logP > 0

logP < 0

logP > 0: the chemical is lipophilic

logP < 0: the chemical is hydrophilic

hydrophilicity and lipophilicity...

help determine the absorption of a chemical and what body tissues a chemical will accumulate

example of logP significance

POPs partition to fat, long half life

logP < 0, lipophilic

toxicokinetics

the quantification of the time course of toxicants in the body during the process of absorption, distribution, biotransformation/metabolism, and excretion/clearance of toxicants

end result of studying toxicokinetic process of a toxicant

is an understanding of the biologically effective dose

absorption

the uptake of an agent from a site of exposure, resulting in an internal dose

distribution

the dispersal of an agent in its original form (unmetabolized) from the site of absorption throughout the body

results in movement of the toxicant from the exposure site to internal areas of the body

metabolism/biotransformation

conversion of chemicals, both those naturally produced within the body (endogenous) and foreign chemicals entering the body (xenobiotics), into different chemical forms, with the goal to make the compounds more water-soluble to facilitate excretion through urine or feces

excretion

the removal of the agent or its metabolites from the body

biological half-life (T1/2)

the time required for the amount of a chemical in the body to decrease to 50% of the starting value

can refer to the entire body burden or within specific tissues

toxicodynamics

the effects of toxicants and their metabolites in biological systems at the molecular, biochemical, and physiological level

the observation of the interaction of the biologically effective dose of the ultimate form of the toxicant with a molecular target

exposure to a toxicant can result in either...

localized effects or systemic effects

localized effects

harmful reactions that occur at the site of contact with a toxic substance

systemic effects

harmful reactions that occur in distant organs or tissues throughout the body, away from the site of contact with a toxic substance

toxicokinetics vs toxicodynamics

toxicokinetics: refers to the "path" an agent takes in a biological system

toxicodynamics: describes the (adverse) effects caused along that path

routes of absorption

dermal

oral

inhalation

other: parenteral, intraperitoneal, intramuscular, intravenous, subcutaneous

a toxicant is only considered to be "internal" once...

it moves across an epithelial cellular membrane that lines an exposure pathway

GI, lung, dermal epithelium

gains entry into the internal fluid compartments of the body

for a systemic effect to occur

the toxicant must defeat barriers to absorption and enter into an internal compartment of the body

otherwise, all toxic responses are confined to the site of exposure, resulting in local effects

when being absorbed by an epithelial cellular membrane,

compounds that are more lipophilic are more easily absorbed

compounds that are uncharged are more easily absorbed

rate of absorption

depends on lipid solubility (logP) and % ionization (pKa + fluid pH) of the toxicant

toxicants move across cell membranes by either

simple diffusion or specialized transport

specialized transport mechanisms

active transport, special transport (facilitated or carrier-mediated diffusion and active transport), endocytosis

primary mechanism of absorption

simple diffusion

factors of net diffusion and diffusion rate

size of molecule

molecular charge and degree of ionization

water solubility

concentration differences across the cell membrane

modes in which a toxicant can cross a cell membrane

simple diffusion

diffusion through protein pores

facilitated diffusion

active transport

endocytosis

exocytosis

percutaneous absorption

absorption from the surface of the skin into the blood of the cutaneous vessels which reside in the dermis

chemicals entering GI tract

must first cross the mucosa somewhere along the tract before gaining entry into the blood

a chemical must first be absorbed in order to exert a systemic toxic effect

degree of absorption through GI

location within the GI tract

pH

time spent in contact with GI tract

physicochemical properties of the chemical

toxicant absorption in the oral cavity and esophagus is low...

because of the relatively short residence compared to the slower transport through the stomach and the gastrointestinal tract

when chemicals are absorbed in the stomach and intestinal tract...

they first pass through the liver circulation before entering into the general circulation of the body, and therefore they cannot escape hepatic metabolism

enterohepatic circulation

a significant amount of toxicants are removed from the venous blood and excreted into the bile, metabolically converted (first-pass metabolism) or stored

unchanged toxicant or its metabolites can be excreted into the bile and back into the small intestine where they may be absorbed or reabsorbed

the wide ranges of pH in the GI system

can dramatically influence absorption

the low pH of the stomach is often less than the pKa of many compounds, causing the compound to be neutral and favoring absorption

respiratory system constitutes a very important route of exposure for...

airborne contaminants

toxic gases, particulates, aerosols, VOS

toxicants can be absorbed in the nasopharyngeal, tracheobronchial, or pulmonary exchange surfaces of the lungs

absorption in respiratory system

lipophilic and low weight gases are quickly absorbed

lipophilicity increases rate of absorption

hydrophilic chemicals decrease absorption with increasing chemical size

chemistry of particulate matter determines retention, absorption, and local/systemic toxicity

pulmonary macrophages

can engulf particulates

some may be cleared into lymphatic system

some may remain within the lungs for an indefinite period of time, as is the case for asbestos and coal dust

material that remains within the respiratory system may produce local toxicity

lung cancer, chronic bronchitis, lung fibrosis, emphysema

the most important parameters when comparing pathways of absorption

the surface area and the thickness of the barrier

comparing pathways of absorption: higher surface area

provides more sites for chemicals to potentially cross, increasing absorption

comparing pathways of absorption: lower thickness

provides a shorter barrier to cross, increasing absorption

bioavailability

a measurement of the extent that a chemical agent reaches the systemic circulation and is available at the site of action

pharmacokinetic studies

used to determine bioavailability via different routes of administration

absolute bioavailability

dose-corrected area under curve of non-intravenous divided by the area under the curve of intravenous

most effective at delivering a systemic dose

IV and inhalation

oral and dermal absorption pathways show

lower peak plasma concentration

delayed absorption

lower bioavailability (less area under the curve)

factors that effect the distribution of a toxicant

lipid solubility

ionization at blood pH

ease of crossing cell membranes

blood flow to the tissue or organ

extent of plasma protein binding

unbound vs bound toxicants

unbound or bound to plasma proteins such as albumin or blood cells

bound toxicants have a reduced potential to enter the cells of the body

only unbound toxicants are able to cross cell membranes

plasma protein binding affects

the distribution of toxicant

the effective dose of the toxicant

the biological half-life of the toxicant

storage of toxicants occurs primarily

in fat and bone, also in kidneys, liver, and connective tissues

lipophilic toxicants are deposited in

fat and adipose tissue

later mobilized back into the blood for further distribution, metabolism, elimination, or redeposition

includes persistent organic pollutants

bone is an important site for

deposition of lead, strontium, and fluoride

apparent volume of distribution (Vd)

the volume of body water throughout which a drug/toxicant "appears" to be distributed

Vd = dose/C0

dose = amount of drug given (mg)

C0 = concentration of drug in blood plasma (mg/mL)

if the toxicant preferentially distributes into tissues

lower concentration of toxicant in plasma (C0)

high Vd

if the toxicant preferentially binds to plasma proteins or remains in the blood

higher concentration in plasma (C0)

low Vd

body burden

a measure of the total amount of toxicant that is harbored within the body, predicted via Vd

X = Vd x C

X = body burden

Vd = apparent volume of distribution

C = concentration in blood

for many lipophilic environmental chemicals knowledge of body burden

is extremely important for estimating exposure for risk assessment calculations

metabolism interchangeable names

biotransformation

drug metabolism

metabolism is affected by factors pertaining to both the toxicant and the host including

existing disease (especially in the liver or enzyme deficiencies), age, sex, genetic variability, enzyme induction, nutritional status

purpose of drug metabolism

make lipophilic toxicants water-soluble so they can be excreted from the body

if toxicants were not metabolized, they would accumulate and reach toxic concentration

key organs in biotransformation

high: liver

medium: lung, kidney, intestine

low: skin, testis, placenta, adrenals

Truck Hitch Trailer: Truck, foreign chemical (xenobiotic)

lipophilic

not charged

not water soluble

poorly excretable

Truck Hitch Trailer: Hitch, phase 1 enzymes

add or expose functional group

still lipophilic

possibly reactive

poorly water soluble

poorly excretable

catalyzed by P450s

Truck Hitch Trailer: Trailer, phase 2 enzymes

conjugate (transfer) endogenous molecules to the functional group

not lipophilic

usually not reactive

water soluble products

excretable

catalyzed by transferases

enzymes

are biological catalysts

provide molecular surface for a chemical reaction to proceed for substrates (reactants)

phase I reactions (functionalization)

oxidation, reduction, hydrolysis, free radical reactions

phase II reactions (conjugations)

major pathways in humans only

glucoronidation, sulfation, acetylation, mercapturation

oxidation reactions

result in the loss of electrons from the parent compound (substrate)

can occur via the removal of hydrogen from the molecule (dehydrogenation)

reduction reactions

result in the substrate gaining electrons

hydrolysis reactions

occur when the toxicant molecule is split into smaller molecules through the addition of water

primary enzyme of phase I enzyme reactions

cytochrome P-450

can catalyze many types of reactions, broad substrate specificity

levels can be increased by chemical exposures, induction

coenzyme required for CPY450

P450 reductase

found where we interface lipophilic xenobiotics: liver, skin, kidneys

enzyme induction

a process in which a molecule (xenobiotic) induces (initiates or enhances) the expression of an enzyme

increases ability to metabolize toxicants

enzyme induction: grapefruit

potent inhibitor of CYP3A4

prevents CYP3A4 from metabolizing target

taking statins with grapefruit results in 12-fold blood concentrations of statin

alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH)

primary routes for ethanol metabolism (two phase I enzymes)

acetaldehyde is toxic intermediate metabolite

people with ALDH21/ALDH22 genotype

frequently experience unpleasant side effects from consuming alcohol due to the reduced ability to break down acetaldehyde

flushing/nausea

Phase II reactions

enzymatic reactions that conjugate the xenobiotic to large water-soluble, charged (polar biomolecules)

functional group (hitch) must be present either in the xenobiotic's original structure or phase I reaction product

Phase II Enzymes/substrate/donor

glutathione-S-Transferase

substrate (trailer): glutathione (GSH)

substrate donor (cofactor): itself

Phase II Enzymes/substrate/donor

glucoronyltransferase

substrate (trailer): glucuronic acid

substrate donor (cofactor): UDP-GA

Phase II Enzymes/substrate/donor

sulfotransferase

substrate (trailer): sulfate

substrate donor (cofactor): PAPS

Phase II Enzymes/substrate/donor

acetyltransferase

substrate (trailer): acetate

substrate donor (cofactor): acetyl CoA