Biochemical Respond
Against Xenobiotic
Introduction
 Xenos: stranger, synonims: biotransformation, drug
metabolism
 Xenobiotic: is compound that foreign to the body that
under normal circumstances is not required by the living
body
 Examples:
pharmaceuticals, pesticides, environmental pollutants
(Pb, CO), industrial chemicals, preservatives, colorings
and flavorings in food products
 Metabolism of xenobiotics are basic to a rational
understanding of pharmacology, therapeutics, pharmacy,
toxicology, management of cancer or drug addiction.
 Xenobiotika compounds can enter the body through:
 Mouth (food, drugs)
 Respiration (cigarette smoke, fumes)
 Skin (pesticide poisoning on farmers)
 Intra venous
 Principal classes of xenobiotics:
 Drugs (antibiotics, analgesic, antipyretic,
supplements
 Carcinogen (food dyes, preservation, alcohol,
nitrosamines, artificial sweetenes)
 Pollutans/environmental chemical (manufactured
environmental chemical)
Xenobiotics Absorption
Xenobiotics
Oral
Intestine
Topical
Skin
Blood
IV
IM, SC, IP
Inhalation
Membranes
Lung
 Effect of xenobiotics:
 the expected effects (the therapeutic effects
of drugs / cure or relieve symptoms of disease)
 unexpected effects (side effects and toxic
effects of drugs)
 Through the process of metabolism and excretion
processes, the body is able to eliminate all
influences that arise after a change in chemical
structure
 Metabolism xenobiotics holds important meaning
in the process of elimination xenobiotics
Metabolism of Xenobiotics
 Xenobiotics metabolism:
 Mechanism of elimination of foreign and
undesirable compounds from the body
 Control of levels of desirable compounds
 Biochemical alteration in the body
 Not detoxication reaction, because:
 Pro drugs
 Pro carcinogenesis
 Metabolism increase biologic activity and/or
toxicity
Divided in 2 phases
 Phase 1: hydroxylation
catalyzed by members of class of enzymes:
monooxygenase or cytovhrome P450s
 Phase 2: conjugation
the hydroxylated or other compounds produced in phase 1
by spesific enzymes, converted to various polar metabolites
by conjugation with glucoronic acid, sulfate, acetate,
glutathion or certain amino acids, or by methylation
Phase I
 Biochemically alter the xenobiotic to change its biologic
effect
 Location:
› Liver (membranes of ER, cytoplasm)
› Other tissues (lungs, intestine, skin, kidneys)
 Enzymes:
› Monooxygenases (hydroxylases, cytochrome P450,
Mixed Function Oxidase/MFO
 Property of enzymes:
› Metabolism of endogenic substances, broad
substrate specificity, inducibility
 Reactions: Hydrolysis, oxidation, reduction
RH + O2 + NADPH + H+  R-OH + H2O + NADP
lipophilic
active
inactive
hydrophilic
inactive
active
 Results:
› Lowering their toxicity
› Increasing their toxicity
› Bioactivation some xenobiotics (ex. procarcinogen 
danger of cell, body damage)
› Increasing their water solubility
Lipophilic
Non polar, hydrophobic
Hydrophilic
Polar
 Poorly soluble in water
 Water soluble
 Need a blood
 Difficult transport
 Freely diffuse through
 Rapidly eliminated with
transporter (albumin)
membranes
 Can be stored in
membranes
 Slowly eliminated from
the body
through membranes
the urine
Cytochrome P450
 Most versatile biocatalyst
 Metabolizes > 50% of all drugs and
chemical in the body
 Membrane-bound protein
 Principal component of active site is the
heme moiety
 NADPH not NADH is involved in the
reaction of mechanism
 Factor affecting metabolism by Cytochrome
P450:
› Diet and nutrition  different nutrient and elemental
›
›
›
›
deficiencies
Hormonal  different hormones affect metabolism
Age and sex  metabolism slower in neonates and
elderly
Genetic  some pathway exhibit genetic variation
Pathological states  diseases involving the liver or
kidney lead to alteration in absorption, distribution
and excretion of xenobiotics
 Inducers of cytochrome P450 synthesis:
› Drugs: barbiturates, steroid, ethanol, nicotine
› Industrial chemical: alcohol, chloroform
› Polyaromatic hydrocarbon: benzopyrenes
 Inhibitors of cytochrome P450 synthesis:
› Competitive binding to and metabolism by
cytochrome P450
› Inhibition of synthesis of heme or cytochrome P450
› Inactivation or destruction of cytochrome P450, or
destruction of the ER by various agent
 Induction of cytochrome P450  important
clinical implication  biochemical mechanism of
drug interaction, example:
Patient is taking warfarin (metabolized by
CYP2C9) and phenobarbital (an inducer P450)
 Level of CYP2C9 will be elevated 3-4 fold after
5 days
 Warfarin will be metabolized much more quickly
 dosage become inadequate  dose must be
increased to be terapeutically effective
Example of a reactions catalyzed by cyt P450
The figure is from: Color Atlas of Biochemistry / J. Koolman, K.H.Röhm. Thieme 1996. ISBN 0-86577-584-2
Some properties of human cytochrome P450s (1)
Involved in phase I of the metabolism of innumerable xenobiotics, including
perhaps 50% of the drug administrated to humans; they may increase,
decrease or not affect the activities of various drugs
Involved in the metabolism of many endogenous compounds (steroid etc)
All are hemoproteins
Often exhibit broad substrate specifity, thus acting on many compounds;
consequently, different P450s may catalyze formation of the same product
Extremely versatile catalysts, perhaps catalyzing about 60 types of
reactions
Basically they catalyze reactions involving introduction of one atom of
oxygen into the substrate and one into water
Their hydroxylated products are more water-soluble than their generally
lipophilic substrates, facilitating excretion
Liver contains highest amounts, but found in most if not all tissues,
including small intestine, brain and lung
Some properties of human cytochrome P450s (2)
Located in the smooth endoplasmic reticulum or in mitochondria
(steroidogenic hormones)
In some cases, their products are mutagenic or carcinogenic
Many have a molecular mass of about 55 kDa
Many are inducible, resulting in one cause of drug interactions
Many are inhibited by various drugs or their metabolic products,
providing another cause of drug interactions
Some exhibit genetic polymorphisms, which can result in atypical
drug metabolism
Their activities may be altered in diseased tissues (eg, cirrhosis),
affecting drug metabolism
Genotiping the P450 profile of patients (eg, to detect polymorphisms)
may in the future permit individualization of drug therapy
 Polymorphism:
Natural variations in a gene,
DNA sequence, or
chromosome that have no
adverse effects on the
individual and occur with fairly
high frequency in the general
population
Structure of human
cytochrome P450 CYP2C9
Some important drug reactions due to mutant or
polymorphic form of enzims or protein
Human CYP families and their main functions. Data adapted from (Gonzalez
1992, Nelson et al. 1996, White et al. 1997, Nelson 1999, Lund et al. 1999)
CYP family
Main functions
CYP1
Xenobiotic metabolism
CYP2
Xenobiotic metabolismArachidonic acid
metabolism
CYP3
Xenobiotic and steroid metabolism
CYP4
Fatty acid hydroxylation
CYP5
Thromboxane synthesis
CYP7
Cholesterol 7α-hydroxylation
CYP8
Prostacyclin synthesis
CYP11
Cholesterol side-chain cleavage Steroid
11β -hydroxylation Aldosterone synthesis
CYP17
CYP19
CYP21
CYP24
CYP26
CYP27
CYP39
CYP46
CYP51
Steroid 17α-hydroxylation
Androgen aromatization
Steroid 21-hydroxylation
Steroid 24-hydroxylation
Retinoic acid hydroxylation
Steroid 27-hydroxylation
Unknown
Cholesterol 24-hydroxylation
Sterol biosynthesis
Phase II
 Phase I derivates are made more polar
(water soluble) through conjugation
reaction
 Location:
 Liver (intestine mucosa, skin): ER, cytoplasm
 Properties:
 Need of an endogenic substance
 Synthetic reactions
 Energy consumption
 Results:
› Highly polar conjugates (increasing water solubility)
› Decreased toxicity
 Important reactions:
 Enzymes: transferase
Conjugation Reactions
 Glucuronidation
Endogen reactant: UDP glucuronic acid
Enzyme: UDP glucuronyl transferase
Location: microsome
Reactive site: OH, COOH, NH2, SH, C-C
Type of substrates: phenol, alcohols, carboxylic
acids, hydroxylamines, sulfonamides
 Examples: acetaminophen, nitrophenol





 Sulfate conjugation
›
›
›
›
›
›
Endogen reactant: phosoadenosyl phosphosulfate
Enzyme: sulfotransferase
Location: cytosol
Reactive site: NH2, OH
Type of substrates: phenol, alcohols, aromatic amines
Examples: aniline, phenol, acetaminophen
 Acetylation
›
›
›
›
›
›
Endogen reactant: acetyl-CoA
Enzyme: n-Acetyltransferase
Location: cytosol
Reactive site: NH2, SO2NH2, OH
Type of substrates: amines
Examples: isoniazid, sulfonamides
 Glutathione conjugation
›
›
›
›
Endogen reactant: glutathione
Enzyme: glutathione S-transferase
Location: cytosol
Reactive site: epoxides, organic halides, organic nitro
compounds, unsaturated compounds
› Type of substrates: epoxides, arene oxides, nitro groups,
hydroxylamines
› Examples: bromobenzene
 Methylation
›
›
›
›
›
›
Endogen reactant: s-adenosyl-methionine
Enzyme: transmethylase
Location: cytosol
Reactive site: NH2, SH, OH
Type of substrates: phenols, amines, catecholamines
Examples: pyridine, histamine, epinephrine
Xenobiotics Drug Metabolism
 Drugs that are active →
phase 1 metabolism
xenobiotik to change
the active drug into
inactive
 Drugs that have not been
active → phase 1
metabolism xenobiotik
convert an inactive drug
becomes active.
Alcohol
METHANOL (CH3OH)






lower narcotic effect than ethanol
slower excretion from the body
metabolized by the same enzymes as ethanol
causes harder sickness (formaldehyde)
serious intoxication: 5 – 10 ml (lethal dose  30 ml)
no symptoms immediately after drunkenness (6 – 30
hours)
 headache, pain in back, loss of sight
 metabolic acidosis
 therapy: ethanolemia  1 ‰ (1 - 2 days), liquids
Ethanol
 A small molecule; both lipid and water soluble
 Readily absorbed from the intestine by passive diffusion
 Small percentage of ingested ethanol (0-5%) enters the
mucosal cells of the upper GI tract (tongue, mouth,
esophagus, and stomach)
 The remainder enters the blood, of which 85 to 98% is
metabolized in the liver, and only 2 to 10% is excreted
through the lungs or kidneys
 Distribution of ethanol in the body:
› the equilibrium concentration of ethanol in a tissue
depends on the relative water content of that tissue
› ethanol is practically insoluble in fats and oils, although
like water, it can readily pass through biological
membranes
› No plasma protein binding ethanol
 Factors affecting ethanol absorption:
› concentration of ethanol, passive diffusion (higher
concentrationgreater absorption), blood flow at site of
absorption
(efficient blood flow greater absorption), rate of
ingestion, food (presence of food in stomach retards
gastric emptying, reduces absorption of ethanol)
 Factors that determine the rate and route of
ethanol oxidation in individuals include:
 genotype  polymorphic forms of ADH and
acetaldehyde dehydrogenase can greatly
affect the rate of ethanol oxidation and the
accumulation
 Drinking history  the level of gastric ADH
decreases and CYP2E1 increase with the
progression from a naïve, to a moderate, a
heavy and chronic consumer of alcohol
 Gender  blood levels of ethanol after
consuming a drink normally higher for women
than men, because of lower levels of gastric ADH
activity
 Factors that determine the rate and route of
ethanol oxidation in individuals include:
 Quantity—The amount of ethanol an individual
consumes over a small amount of time
determines its metabolic route.
Metabolism of Ethanol
 Metabolism occurs by two pathways
 The first pathway comprises two steps
 The first step, catalyzed by the enzyme alcohol
dehydrogenase, takes place in the cytoplasm:
 The second step, catalyzed by aldehyde
dehydrogenase, takes place in mitochondria:
First Pathway
 Ethanol consumption leads to an accumulation of
NADH
 High concentration of NADH inhibits
gluconeogenesis by preventing the oxidation of
lactate to pyruvate  will cause the reverse
reaction to predominate, and lactate will
accumulate  the consequences may be
hypoglycemia and lactic acidosis
 The NADH glut also inhibits fatty acid oxidation, the
excess NADH signals that conditions are right for
fatty acid synthesis  triacylglycerols accumulate
in the liver, leading to a condition known as "fatty
liver"
Second Pathways
 The ethanolinducible microsomal ethanol-oxidizing system
(MEOS)
 Cytochrome P450-dependent pathway generates
acetaldehyde and subsequently acetate while oxidizing
biosynthetic reducing power, NADPH, to NADP+
 Because it uses oxygen, this pathway generates free radicals
 damage tissues
 Because the system consumes NADPH, the antioxidant
glutathione cannot be regenerated  exacerbating the
oxidative stress
 Approximately 10 to 20% of ingested ethanol is oxidized
through a microsomal oxidizing system (MEOS), comprising
cytochrome P450 enzymes in the endoplasmic reticulum
(especially CYP2E1)
Figure 1 Oxidative pathways of alcohol metabolism. The enzymes
alcohol dehydrogenase (ADH), cytochrome P450 2E1 (CYP2E1), and
catalase all contribute to oxidative metabolism of alcohol. ADH, present
in the fluid of the cell (i.e., cytosol), converts alcohol (i.e., ethanol) to
acetaldehyde. This reaction involves an intermediate carrier of
electrons, nicotinamide adenine dinucleotide (NAD+), which is reduced
by two electrons to form NADH. Catalase, located in cell bodies called
peroxisomes, requires hydrogen peroxide (H2O2) to oxidize alcohol.
 Liver mitochondria can convert acetate into
acetyl CoA in a reaction requiring ATP
 The accumulation of acetyl CoA has several
consequences:
› ketone bodies will form and be released into the
blood, exacerbating the acidic condition already
resulting from the high lactate concentration
› If ethanol is consistently consumed at high levels, the
acetaldehyde can significantly damage the liver 
leading to cell death
 Liver damage from excessive ethanol
consumption occurs in three stages
 the aforementioned development of fatty liver
 alcoholic hepatitis groups of cells die and
inflammation results
 cirrhosis fibrous structure and scar tissue are
produced around the dead cells
 The cirrhotic liver is unable to convert
ammonia into urea, and blood levels of
ammonia rise  toxic to the nervous system
 cause coma and death
 Cirrhosis of the liver arises in about 25% of
alcoholics, and about 75% of all cases of
liver cirrhosis are the result of alcoholism
• Alcoholic Liver Disease (ALD) is a term used to describe the
spectrum of liver injury associated with acute and chronic
alcoholism
• The 3 stages of Alcoholic Liver Disease are:
• Hepatic steatosis (fatty change)
• Alcoholic hepatitis
• Alcoholic cirrhosis
• Interrelationships among stages of Alcoholic Liver Disease:
Some Metabolic Consequences of the increased
NADH/NAD ratio
Metabolic Effect
Clinical Correlate
Decreased gluconeogensis
Hypoglycemia
Increased Lactate
Elevated blood lactate Possible gout
Possible increased ketoacid
Decreased Krebs (TCA) cycle
Ketoacidosis
Inhibition of most oxidations that use liver NAD
Inhibition of some drug and hormone metabolism
Decreased fatty acid oxidation and increased fatty
acid synthesis
Fatty Liver
Elevated blood Lipids
Alcohol, biochemistry and metabolism, Ghassan Hemased, MD
Acute Ethanol Toxicity
Ethanol
ADH
MEOS
Inter polate into
membranes
Mem.fluidity
Toxicity effects(Brain)
Acetaldeyde
Adducts with proteins
+ nucleic acid
ADH
ed
NADH/NAD+
Acetate
1. Lactate/Pyruvate
Acetyl COA
2. Gluconeogensis
F.A Synthesis
3. F.A oxidation
Fatty Liver
4. Glycophosphate
Dehydrogenase
leading to
glycrophosphate
Alcohol, biochemistry and metabolism, Ghassan Hemased, MD
Potential alcohol-medication interactions involving cytochrome P450
enzymes in the liver (Alcohol Research and Health, 1999)