Basic & Clinical Toxicology (1801446-3)

Lecture:3

Toxicokinetic: Biotransformation of
toxicants
Dr Hanouf Bafhaid
hsbafhaid@uqu.edu.sa
D e p a r t m e n t o f P h a r m a c o l o g y a n d To x i c o l o g y
College of Pharmacy
Umm Al-Qura University
Learning outcome
At the end of this lecture, you will be able to:

Identify the key compartments that determine the fate of toxicants in the body

Explain the different transports system of toxicants and factors affecting absorption

Demonstrate the factors affecting the toxicants distribution

Discuss the principles of toxicant metabolism

Illustrate the metabolism pathways and the enzyme reactions

Define the several routes of toxicants excretion

 Toxicokinetics
What is the fate of toxicants that enter the body?

A: route of absorption, transport across membrane, factors affecting absorption

D: plasma protein, volume of distribution, tissue localisation

M: phase I and II metabolism, enzyme inducers and inhibitors

E: routes of toxicants excretion, factors affecting excretion

Figure 1. Toxicokinetic processes which control the fate

of toxicants within the body
 Absorption
The process of transportation of unchanged toxicants from the site of administration to the body circulation system

Absorption pathways:
-Gastrointestinal, respiratory, and dermal systems
-These systems are lined with epithelia that present significant
barriers to the entry of foreign substances due to tight junctions
between their cells or continuous lipid layers in the case of skin

Types of transport across membranes: (fig.2)

1-Passive transport (simple diffusion, facilitated diffusions,
filtration)
2-Active transport (require energy) can become saturated

Figure 2. Processes of the transport of toxicants across membranes

 Absorption
Attributes of absorbed molecules

1-Size
-Molecules with a molecular mass of 500 Da or less penetrating cell membranes in a process
known as passive diffusion (transcellular permeability)
-Small molecules (150 Da or less) that are highly water soluble may enter tissues via filtration
(paracellular permeability)

2-Solubility (molecules hydro/lipophilicity)

- -rich, nonaqueous organic
barrier
-Hydrophilic molecules dissolve poorly in a lipid environment. Such molecules often possess
heteroatoms (e.g. N, O) or polarised functional groups

3-Charge (molecules ionisation status)

-Neutral (uncharged, nonionic) molecules passively cross lipid bilayers of the epithelial
membrane
-Charged molecules can not cross lipid membrane
-The pKa of the molecule dictates its ionisation behaviour within a particular pH environment

Figure 3. Key properties influencing membrane penetration by

xenobiotics
 Absorption
-Molecules entering the body require an ability to dissolve in hydrophobic, nonpolar organic solvents to traverse the lipid-rich
-tract or lung airways

-A significant pH gradient exists throughout the gastrointestinal tract (e.g. from pH 1 2 in the stomach to pH 5 6 in the duodenum and
pH 8 in the ileum and colon), the ionisation state of ingested molecules frequently changes as they transit through this organ.

-Majority of toxicants absorption occurs in zones where the molecule assumes a neutral pH, for most molecules the duodenum remains
the main site of GI-tract absorption due to its large surface area and high blood flow

-Acidic compounds are (i.e. low pKa) nonionised within the acidic gastric environment. Basic compounds are nonionised within the
neutral pH environment prevailing within the small intestine
 Distribution
A process by which toxicants move throughout the body and reach their site of action

-Xenobiotics may distribute into any or all of the fluid compartments: plasma, interstitial fluid and intracellular fluid

Volume of distribution:

Vd (L) = administered dose (mg) / plasma concentration (mg/L)

High Vd distributed to all body fluid, low plasma concentration
Low Vd limited to plasma concentration

Tissue specific accumulation:

Aminoglycoside kidney (toxicity)
Paraquat lung Figure 4. The distribution of absorbed toxicants within the body. The shading
intensity indicates toxicant concentrations in the respective compartments
DDT adipose tissue
Heavy metal bone
Tetracycline bone and teeth (teeth discoloration, impaired bone growth)
 Distribution
Factors that influence distribution:
1. Physicochemical properties of compound: molecular size, lipophilicity
-Lipophilic toxicants penetrate deep into body tissues usually leave negligible concentrations within the bloodstream
-Hydrophilic toxicants display a preference for the blood compartment during the distribution phase. They achieve high
concentrations within the proximal tubules and excreted
2. Special compartmental and barriers, e.g., blood brain barrier, placental barrier
3. Plasma protein binding affinity
-Molecules bound to plasma proteins such as albumin (high the capacity to bind many compounds)
-Molecules strongly bound to plasma protein will have a long duration of action
-Mostly non-covalent-binding i.e. reversible
-Consequence decrease free compound, increase plasma half-life, saturation of binding sites
 Metabolism
The metabolism of foreign compounds is to convert fat soluble compounds into water soluble forms

The roles of metabolism are to render compounds:

-life
this leads to

This is accomplished by converting lipid-soluble (nonpolar), no excretable xenobiotics to polar, water-soluble

compounds accessible for elimination in the bile and urine.
 Metabolism
What are the functions of biotransformation?
1- Conversion of an active compound to less active (inactivation) e.g. phenytoin p-hydroxphenytoin
2- Conversion of an active compound to more active metabolite(s) (bioactivation) e.g. codeine morphine
3- Conversion of an inactive compound (pro-drug) to active metabolite(s) (activation)
e.g. levodopa dopamine
4- Conversion of an active compound to equally active metabolite(s) (no change in the activity)
e.g. digitoxin digoxin
5- Conversion of an active compound to active metabolite(s) having new toxicological activity (change in activity)
e.g. iproniazid (antidepressant) isoniazid (tuberculosis)
 Metabolism
Many tissues express enzymes to mediate toxicants metabolism including kidney, liver, blood, gut, skin (liver is the
major detoxification organ)

The xenobiotic metabolizing enzymes can be divided into two main groups:
1. Non-microsomal enzymes present in the cytoplasm, plasma, and mitochondria and occur mainly in liver and GI tract
These enzymes catalyse few oxidative, hydrolytic and conjugative reactions
2. Microsomal enzymes are present in the smooth endoplasmic reticulum of liver and other tissues and classified into
phase I and II metabolism
 Metabolism
Xenobiotic metabolism is classified into 2 types:
Phase I: increase polarity
Function: adding or exposing a functional group
Reactive group: -OH, -SH, -NH2, or -COOH
Reaction: oxidation, reduction, hydrolysis
Enzyme: CYP450, flavin monooxygenases (FMO), hydroxylase, alcohol and aldehyde dehydrogenase, carboxylesterases
Activity of metabolite: variable (increase or decrease)

Phase II: increase hydrophilicity, water solubility

Function: catalyse conjugation of xenobiotics or their phase I metabolites and endogenous substrates
Reaction: conjugation: glucuronidation, sulphation, acetylation, methylation
Enzyme: sulfotransfrase, glutathione s-transferase, glucuronosyl transferase
Activity of metabolite: mostly inactivation, some conversion to more toxic metabolite
 Metabolism
CYP450
-Cytochrome P450 s are super family of haem-containing mono-oxygenase
-Collection of isoenzymes, possessing a haemoprotein (cofactor)
-Catalyse oxidation of a wide range of substrates:
Endogenous: steroids, vitamins, lipids
Exogenous: drugs, environmental contaminants
-Oxidative reaction requires:
CYP450 haemoprotein, NADPH, CYP450 reductase, O2

-Major human CYP isoenzymes: Figure 5. Estimate of CYP isoenzymes contribution to xenobiotic
metabolism Copyright (2008, American Chemical Society)
CYP3A4/5 is most important during the metabolism of drugs
CYP2E1 metabolises many synthetic chemicals of relevance to toxicology
 Metabolism
Phase I reaction
Table 1: Types of phase I biotransformation reactions (oxidation) with examples
A. Oxidation: it is the most common reaction, proceed by adding oxygen to a foreign compound/by removing hydrogen atoms from a
molecule add functional group,
Types of reactions functional modification Reaction (example)

1- N-/O-/S-dealkylation Remove alkyl R-O-C2H5 R-OH + CH3CHO

(e.g.CH3-)
2-Hydroxylation Aliphatic hydroxylation Heterocyclic ring hydroxylation
Aromatic hydroxylation OH
R-CH2-H R-CH2-OH
Heterocyclic ring hydroxylation Add hydroxyl Aromatic hydroxylation
N-hydroxylation (-OH) N-hydroxylation
Aliphatic hydroxylation
R-NH2 R-NH-OH

3-N-/S-oxidation Oxide formation R-NH2 RNH -OH

4-Desulfuration Remove sulfar RSH ROH

5-Deamination Remove amine R-CH2NH2 R-CHO + NH3

 Metabolism
Phase I reaction

Table 2: Types of phase I biotransformation reactions (reduction and hydrolysis) with examples
B. Reduction: is acceptance of one or more electrons(s) from another substrate by the addition of extra
hydrogen atoms to a molecule (or the removal of oxygen atoms) mask a functional group

Types of reactions Modification Reaction (example)

1-Azo reduction Add hydrogen molecule R-N=N-R' R-NH2 + R-NH2
2-Nitro reduction Remove oxygen R-NO2 R-NH2
C. Hydrolysis: is the process of cleaving of a foreign compound by the addition of water mask a functional
group
Types of reactions Modification Reaction (example)
1- amides Add water molecule RCONR R RCOOH + HNR
2- esters Add water molecule RCOOR RCOOH + R OH
 Metabolism
Phase II reaction
-In many cases, phase I metabolism is not sufficient for excretion so further metabolism is required
-Phase II is synthetic and involve linking compound with a conjugate
-Most pathways require energy (ATP)
Phase I Phase II
-X X-OH X-O-conjugation

Table 3: Types of phase II biotransformation reactions (conjugation)

Conjugation: foreign chemicals linked to a hydrophilic endogenous substrates present in the liver

Types of reactions Functional Groups of Xenobiotics Conjugate (add)

Glucuronidation -OH, -COOH, -NH2, -SH Glucuronic acid
Methylation -OH, -NH2, -SH Methyl group (-CH3)
Acetylation NH2, -SO2 Acetyl group (-COCH3)
Glutathione conjugation -F, Cl, -Br, electrophiles Glutathione (GSH)
Sulfonation -OH, -NH2 Sulfonate (-SO3-)
 Bioactivation and tissue toxicity

-The bioactive metabolites interact with the body tissues cause toxicities such as carcinogenesis, teratogenesis, and tissue necrosis.
-The bioactivation reactions are generally catalysed by cytochrome P450- dependent mono-oxygenase systems, some other enzymes are
involved e.g. those in intestinal flora
-The reactive metabolites primarily belong to three main categories: electrophiles and free radicals

-Electrophiles Molecules which are deficient in electrons pair with a positive charge, react by sharing electron pairs with electron-rich
atoms in nucleophiles
-Free radicals molecules contain one or more unpaired electrons (odd number of electrons) in their outer orbit
-Both electrophiles and free radicals irreversible binding with proteins, nucleic acids, and lipids cause damage to DNA and protein, lipid
peroxidation and carcinogenesis

Compound Active metabolite Toxic effect

Carbon tetrachloride Trichloromethyl radical Lipid peroxidation

Methanol Formaldehyde Ocular toxicity

Chloroform Phosgene Hepatic necrosis
 Enzyme inducers
-Xenobiotics may increase activity of certain liver microsomal enzymes (CYP450) phase I
-Xenobiotics interact with DNA and increase the synthesis of microsomal enzyme proteins
-Some compounds may stimulate their own metabolism (self-induction)
-It requires repetitive administration of the inducing agent over a period of several days and, once the induction started, it may continue
for several days
-Increase metabolism decrease activity of other drugs or increase the formation of toxic metabolites

Inducers Drug affected

Barbiturates Warfarin
Phenytoin Quinidine
Carbamazepine Theophylline
Rifampicin Cyclosporin
Smoking Calcium channel blockers
Chronic alcoholism
 Enzyme inhibitors

-Xenobiotics may decrease the activity of liver microsomal enzymes (CYP450) phase I
-The administration of hepatotoxic agents causes either an increase in the rate of enzyme degradation or a decrease in
the rate of enzyme synthesis
-Decrease metabolism increase activity or toxicity of drugs

Inhibitors Drug affected

Azole antifungals Warfarin
Cimetidine Quinidine
Macrolides Phenytoin
Quinolones Theophylline
Valproic acid Benzodiazepines
 Polymorphisms in CYP Pathways
-
susceptibility to toxicants

-Changes in the genomic DNA sequencing of the enzyme can cause:

No effect on enzyme activity
Increase enzyme activity (by gene duplication)
Abolish enzyme activity (by gene deletion, alter splicing)
Alter enzyme activity (by change in structure) cause changes in substrate specificity

-CYP2D6 polymorphisms
Patient subgroups that display exaggerated responses to the cardiovascular drugs debrisoquine.
The inability to metabolise debrisoquine was linked to a 2D6 polymorphism that was found to vary in its
prevalence in different ethnic groups (e.g. 5 , while the incidence in
Asian populations is ~ 1 %)
 Excretion
A process by which toxicants and/or their metabolites are irreversibly transferred from body to external environment
-Route of excretion: renal, bile, lung, enterohepatic circulation
-Other routes of excretion: sweat, saliva, breast milk

The rate of excretion is dependent on:

Plasma half-life
Elimination rate constant (first order kinetic)
Biological half-life
Kidney damage
Insufficient liquid intake

Elimination kinetics
First order kinetics (majority of drugs): rate of elimination is directly proportional to drug concentration (Clearance
remain constant)

Zero order kinetics - linear (Few of drugs): rate of elimination remains constant irrespective of drug concentration
(clearance decreases with increase in drug concentration)
 Excretion
1-Renal excretion
Renal excretion occurs by three different mechanisms:
1-Glomerular filtration (most xenobiotics): unbound molecules are filtered in this manner
2- Active tubular (carrier mediated process) secretion of ionized substances in the proximal tubules
3- Passive tubular reabsorption of unionized substances can be altered by changes in the pH

Figure 6. Mechanisms of renal excretion

 Excretion
2-Biliary excretion
-The ability of the liver to remove chemicals via bile is important to bodily defences against large, lipophilic molecules,
conjugated metabolites formed from bulky molecules
-The biliary route of excretion plays a major role in the elimination of anions, cations, and nonionized molecules containing
both polar and lipophilic groups

Biliary excretion:
Drugs are actively transported into bile
Drugs must be above the M. wt. of 500 Da to be excreted

3-Enterohepatic circulation
Substances that are metabolized in the liver, excreted into the bile, and passed into the
intestinal; they are reabsorbed across the intestinal mucosa and returned to the liver via portal
circulation

Consequence:
-Pharmacological activation/inactivation of compound
-Toxicological activation
-Drug interaction
Figure. 7 Enterohepatic circulation process
References
Goldfrank's Toxicologic Emergencies, 11th edition, 2015 by Lewis S. Nelson

An Introduction to Toxicology, 2014 by Philip C. Burcham

Fundamentals of Toxicology Essential Concepts and Applications, 2016 by P.K. Gupta

Principles of Biochemical Toxicology, 4th edition, 2009 by John A. Timbrell