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LECTURE 3. Toxic action PHASES.

 
Exposure phase. 
Pathways for xenobiotics  
Pathways for xenobiotics. 
ABSORPTION. 
DISTRIBUTION. 
STORAGE OF TOXIC SUBSTANCES. 
EXCRETION.

Substance  organism  Penetration Effect


Phases of toxic action The processes suffer from the toxic which is
brought into contact with a living organism to which exert their action

• Exposure phase: Situation in which a


substance may influence on an organism

•Toxicokinetic PHASE (Availability


biological
g action):
) absorption,
p , distribution,,
Fixation-biotransformation and excretion.

•Toxicodynamic
Toxicodynamic PHASE : Toxic Action
substance by interaction with tissue / organ target

• Toxic or contact local action.


• Systemic toxic action
LECTURE 3
T i l i l Paradigm
Toxicological P di Toxicodynamics
Toxicokinetics
What We do to the Chemical Wh t the
What th Chemical
Ch i l DDoes tto U
Us

Biologically Early Altered


Internal
Exposure Effective Biological Structure & Disease
Dose
D
Dose Eff t
Effect F
Function
i

Absorption Susceptibility and


Distribution Modifying Factors
Metabolism (Genetics and Nutritional Status)
Excretion
Storage
LECTURE 3

S t i  t i   ti  T i ki ti
Systemic toxic action: Toxicokinetics
The study of the movement of toxic species within the body is known as
toxicokinetics.

Exposure Absorption Distribution and Elimination


metabolism
AIR INHALATION URINE

WATER INGESTION FECES.


TARGET
PLASMA
FOOD ABSORPTION ORGAN AIR
SKIN EXPIRED

OTHER OTHER OTHER

The toxicokinetic phase encompasses absorption of toxicants into the organism and all
processes which follow, transport by body fluids, distribution and accumulation in
tissues and organs, biotransformation to metabolites and elimination (excretion) of
toxicants and/or metabolites from the organism.
LECTURE 3
THE TRANSIEN THROUGH THE BODY DEPENDS ON
The ability of the xenobiotic to cross cell membranes
The absorption, distribution, and excretion of xenobiotics involves passing
through various cell and organ membranes.

• Skin, lungs and GIT may be considered as


barriers separating higher organisms from the
environment.

phospholipid
bilayer
y

LECTURE 3

1. ABSORPTION

Process whereby chemicals cross body membranes and enter blood


(INHALATION, INGESTION, SKIN ABSORTION, OTHERS)
Absorption occurs when a substance passes into a cell or into the
circulatory system regardless of the route of exposure.
Absorption mechanisms

LECTURE 3. Absorption
Absorption
p mechanisms

CONCENTRATI
PROCESS MECHANISM PRESSURE ON GRADIENT
GRADIENT. CONDITIONS
DEPENDENT
Passive Dilution Not Yes 1. Lipid (distribution
diffusion coefficient lipid / water)
2 Size (Mw)
2.
3. Degree of ionization
(pH and pKa)*

Filtration Pores step Yes Yes Molecular size


Substance <pore
Facilitated Transporter Not Yes No Energy required
transport
Active Carrier + ATP Not Not Structural Analogy
transport Energy Expenditure
The system may be
saturable
Competitive process and
unidirectional

* The pH at which an acid is 50% dissociated (ionized = nonionized) is called pKa.


The degree of ionization of the chemical depends on its pKa and the pH of the solution.

LECTURE 3. Absorption

Absorption mechanisms

Absorption  Distribution  Fixing: Passive diffusion


Absorption, Distribution, Fixing:

Renal excretion:
• Filtration Filtration soluble substance
• Reabsorption: Passive diffusion and Active 
Transport

Biliary excretion: Passive diffusion and Active Transport

LECTURE 3. Absorption
P
Passage of a xenobiotic from the outside to the biological fluids
  f    bi ti  f  th   t id  t  th  bi l i l fl id
Gastrointestinal tract, respiratory system and skin

A) Digestive tract

–Absorption of toxic substances in the


digestive tract occurs primarily in the
small intestine.
pH Absorption
Stomach 1-3 weak acids
Duodenum 5-7 weak bases
Colon 7-8 weak bases

By Passive diffusion

LECTURE 3. Absorption

A) Digestive tract

FACTORS AFFECTING THE UPTAKE


• pH digestive tract
• enzymes stomach or bowel
• intestinal microbiota
Characteristics of • surface area
digestive tract • permeability
• intestinal motility
• gastric emptying
• food / drug

• liposolubility
p y
Physico-chemical • size (PM)
properties of
substances
• similarity with endogenous molecules
• ionisation of the same or the electric charge
LECTURE 3. Absorption
A) Digestive
Di ti tract
t t

Oral Level

Oral absorption (PH = 7) for Passive diffusion
alcohols  nitroglycerin  cocaine  steroids  etc
alcohols, nitroglycerin, cocaine, steroids, etc..

Esophageal Level
Very little absorption occurs
Chemical burns or irritation

Stomach and intestine level


Passive diffusion
• Chemical structure of the substance
• Ionization of the substance depends on the pKa and pH of the
digestive tract.

LECTURE 3. Absorption

A) Digestive
Di ti tract
t t
Henderson--Hasselbalch Equations
Henderson Equations..
The pKa of a weak acid or weak base is the pH at which there are equal
amounts of the protonated form and the unprotonated form. The Henderson-
Hasselbalch equation can be used to determine the ratio of the two forms

Weak acid: For weak acids, the protonated form is nonionized.


[AH] Ka [A-] + [H+]
Ka= [A-] [H+] / [AH]
[AH] = unionized form
pH
H = pK L [A-] / [AH]
Ka+ Log [A-] = Ionized form

pH-pKa= Log [A-] / [AH]


pH = pK ……..….[A-] = [AH]
10pH-pK
H K = [A-] / [AH]
pH> pKa………… [A-]> [AH]
pH <pKa….…..…. [A-] <[AH]

LECTURE 3. Absorption
A) Digestive
Di ti tract
t t
Weak base
[BH+] Ka
For weakk bases,
F b the
h protonated
d
[B] + [H+] form is ionized.

Ka= [B] [H+] / [BH+]


[B] = ionized form
pH = pKa+ Log [B] / [BH+] [BH+] = Ionized form
ppH-pK
p a= Logg [B] [ +]
[ ] / [BH

pH = pK ……….[B] = [BH+]
10pH-pK = [B] / [BH+] pH> pKa ……….[B]> [BH+]
pH <pKa………. [B] <[BH+]

LECTURE 3. Absorption

A) Digestive
Di ti tract
t t
Stomach pH = 1
Phenobarbital (weak acid) pKa = 77.22
22
pH-pKa= Log [A-] / [AH]
1-7.22 = log [ -] / [AH]
g [A [ ]
-6.22 = Log [A-] / [AH] ..................... 10 -6.22 = [A-] / [AH]
If [HA] = 1 10 -6.22 = [A-]
The predominated form at pH 1 is nonionized phenobarbital therefore it will
be well absorbed in the stomach.

The stomach, which has high acidity (pH 1-3), is a significant site for
absorption of weak organic acids, which exist in a diffusible, nonionized and
lipid-soluble form. In contrast, weak bases will be highly ionized and
therefore poorly absorbed.

LECTURE 3. Absorption
A) Digestive
Di ti tract
t t
Aniline (weak base) pKa = 5
pH-pK L [B] / [BH+]
H Kto= Log
1-5 = log [B] / [BH+]
-4 = Log [B] / [BH+] ............................ 10 -4 = [B] / [BH+]

If [BH+] = 1 10 -4 = [B]
Ionized aniline predominates at pH 1,
1 therefore it is not practically
absorbed in the stomach.

The intestine, particularly in the small intestine is near neutral


(pH 5-8), both weak bases and some weak acids are
nonionized and are usually readily absorbed by passive
diffusion.
Lipid soluble, small molecules effectively enter the body from
the intestine by passive diffusion

LECTURE 3. Absorption

A) Digestive
Di ti tract
t t

Enterohepatic
circulation

Biliary excretion: MW> 300; conjugate Compounds,


Compounds polar,
polar nonpolar,
nonpolar
non-ionized, cationic, anionic

Active Transportation with


ith specific transporter
LECTURE 3. Absorption
TOXIC SUBSTANCES ABSORPTION
Gastrointestinal Tract depends on
on: T
Type
pe of cells
the length of time that a compound is available for absorption
pH in the stomach and intestine

•Poor absorption
•Exceptions: nicotine and nitroglycerin

•Poor absorption.
absorption Weak Bases
•High absorption. Weak acids
•Chemical breakdown of some substances
•Presence of food

•Large surface area


•Greater absorption Slightly dissociated acids
anddb
bases
•Passive diffusion, Facilitated and active
transport
•Slow movement of substances
•Microflora
Microflora and enzymes that affect the toxicity
Very little absorption of toxic substances (carcinogenic nitrosamines)
occurs in the large intestine.
LECTURE 3. Absorption

B) RESPIRATORY TRACT
• Route of entry and exit of substances
• Toxicity contact: irritating, caustic or corrosive (NH3, NOx, SOx)
• Target organ of toxic blood (paraquat)
FUNCTIONS • Biotransformations
• Accumulate lipophilic compounds, amines, ethanol

• in favor of their concentration gradient


•Great alveolar surface contac (100 m2 )
FAST AND • Excellent irrigation (80 m2, High blood perfusion)
EFFECTIVE • Extremely thin alveolar epithelium separate the inhaled
air from the blood stream.
DIFFUSION
A gas or vapor simple passive diffusion. Blood solubility is a major factor in determining the
rate of absorption
• Water-soluble gases and liquid aerosols (high solubility in the blood) the rate of
transfer depend on respiratory rate.
•Poor water soluble gases or vapors, blood can become quickly saturated. The rate of
transfer depend on blood flow.

LECTURE 3. Absorption
B) RESPIRATORY TRACT
Inhalation
•The absorption of solid particles, regardless of solubility, is dependent upon
particle size
Large particles are deposited in the nose -
little absorption.
* Small p particles p
penetrate to the alveoli
and can be absorbed.
* Some particles remain in the alveoli
indefinitely. coal dust and asbestos fibers
lead to disease ((ex. asbestosis). )

Both the entry of airborne toxicants into the circulatory system and the distribution to the
body tissues depend upon several factors:
• concentration of the toxicant in the air
• so
solubility
ub ty of
o tthe
e toxicant
to ca t in tthe
ebblood
ood a
andd ttissue
ssue
• the toxicant’s molecular or particular size
• respiration rate
• duration of exposure
• functional integrity
g y of the respiratory
p y tract ((chronic bronchitis,, alveolar breakdown ((emphysema),
p y ),
fibrotic lung disease, and even lung cancer)

LECTURE 3. Absorption

c) SKIN
The skin is is relatively impermeable to most ions as well as aqueous solutions

Epidermis
The outermost layer of cells packed with keratin is called
stratum corneum (cornified layer).

the epidermis has a impermeable characteristic. Do not


contain blood vessels

Dermis
–composed primarily of connective tissue.
–Blood vessels are distributed throughout
g the dermis
Also found hair follicles, sweat glands, and sebaceous
glands.

The ability of a toxic substance to penetrate the stratum corneum determines the rate at which
the substance will be absorbed by the skin.
Toxicants move across the stratum corneum by passive diffusion.
Small amounts of chemicals may
y be absorbed through
g the sweat g
glands, sebaceous
glands, and hair follicles.

LECTURE 3. Absorption
c) SKIN
• Factors that influence the absorption of toxicants:

• Damage
D t the
to th integrity
i t it off the ki Abrasion,
th skin Abrasion scratching,
scratching or cuts to the skin
Some acids, alkalis, and corrosives
Burns and dermatitis.

• Hydration state of a the stratum corneum


Polar compounds (which are water-soluble) low diffusion through the surface of
the hydrated keratinized layer
layer. Moisturizing can increase penetration
penetration.

Nonpolar compounds (which are lipid soluble) dissolve in and diffuse through
the lipid material between the keratin filaments

• Solvent as carrier
O
Organic
i chemicals
h i l di dissolved
l d iin water
t d do nott easily
il penetrate
t t ththe skin
ki
Organic solvents, are lipid-soluble and therefore penetrate the cell
membrane readily.

• Molecular size
LECTURE 3. Absorption

2. DISTRIBUTION

The distribution, toxicokinetics second phase, is the passage of the poison from
the blood to the different tissues which exert their action or either accumulate.

LECTURE 3. Distribution
2. DISTRIBUTION
Major distribution of an absorbed chemical is by blood with only
minor distribution by lymph.

An adult must ≃6L blood. Cardiac


output is 6L/min

At 1 min the blood has traveled


throughout the body

LECTURE 3. Distribution

2.1. Apparent volume of distribution (VD)


Xenobiotics can be distributed in volumes higher than physiological.

"Total
Total body fluid that would be needed to contain a
toxicant in the body, if it were water soluble and
uniformlyy distributed"

VD = Qt/ Ct Qt = VD· Ct

VD= Volume of distribution


Qt = Total amount of a xenobiotic at time t in the body (mg)
Ct = Plasma concentration at time t (mg / L)

non-polar substances, low rates of ionization or low plasma binding: VD


polar substances, highly ionized and high plasma binding : VD

LECTURE 3. Distribution
2.2. Factors influencing the distribution:

1)Route of exposure

2)The amount of blood flowing through the tissue.

3) Permeability of blood vessels for the xenobiotic

4)Storage of chemicals in tissues


.

LECTURE 3. Distribution

2 2 Factors influencing the distribution:


2.2.

1) Route of exposure
►Chemicals absorbed through the GIT are
carried directly to the liver
liver, then to the heart
heart,
the lungs then to other organs. “First pass
effect.“(intraperitoneal)
Ej: biotransformation of the propranolol
(cardiac depressant) is about 70% when
given orally.

►Chemicals absorbed through the skin or


lungs enter the blood, then go to the heart
g
and then are distributed to various organs
before they reach the liver. (intravenously or
intramuscularly)
The blood level of a toxicant not only depends on the site of absorption but
also the rate of biotransformation and excretion.
LECTURE 3. Distribution
2.2. Factors influencing the distribution:
2)The amount of blood flowing through the tissue

Over 60% of cardiac 
output goes to well‐
Those tissues with increased blood supply will be perfused organs such 
more likely to incorporate the toxic from the as liver and lungs.
bloodstream
LECTURE 3. Distribution

2.2. Factors influencing the distribution:


3) Permeability of blood vessels for the xenobiotic

Blood vessel Tissue Cell


Cell Membrane
Vascular Wall
I t
Intracellular
ll l Space
S

Space intracorpuscular
Space
Interstitial
Intravascular space

Intracellular Membranes Components

The variability of blood vessels due to the pores of the cell


membrane junctions.

Example: Skeletal muscle has 00.2%


2% endothelial junctions
junctions.
Lipophilic substances pass through the blood vessel better than
hydrophilic. LECTURE 3. Distribution
Types of capillaries
Structure:
With porus: 
Discontinuous endothelium  endothelium  Continuous or endothelium  Continuing glial cells 
and basement membranes 
db b with closed spaces or basement membrane 
b b L
Layered unbound
d b d
with many spaces on the basement  have spaces
membrane

A B C D

Location:
Liver, spleen and Choroid plexus (eye),  H t
Heart muscle,
l Spinal cord,
marrow gastrointestinal mucosa, smooth and  brain
kidneys and glands skeletal muscle

Hydrophilic molecules permeability


Very good Good Poor Virtually 
impermeable
p

LECTURE 3. Distribution

2.2. Factors influencing the distribution:


3) Permeability of blood vessels for the xenobiotic

Substances in interstitial fluid must through


g numerous membranes

CNS:
P
Penetrate:
t t liposoluble
li l bl substances
b t
Very slowly hydrosoluble and ionized
No pass: compounds linked to plasmatic proteins

Blood-brain barrier: astrocytes possess many small branches, which form a


barrier between the capillary endothelium and the neurons of the brain
brain. Effective barrier;
vascular membrane with very fine pores, thick endothelium and few transport proteins.

In children: Pb causes encephalitis.

Placental barrier: liposoluble substances accumulate in fetus


(diffusion passive,facilitated diffusion and active transport).
F
Fetus: accumulation
l i and d toxicity
i i more lilipophilic
hili than
h the
h mother.
h

LECTURE 3. Distribution
2.2. Factors influencing the distribution:
4)Storage of chemicals in tissues
Storage of chemicals in tissues
g of chemicals within the body
Main sites for storage y are:
• Plasma proteins
• Adipose tissue (fat) Acidic drugs are bound to the most
• Bone abundant plasma
• Liver and kidneys i ((albumin);
protein lb i ) while
hil bbasic
i d
drugs bi
bind
d to
-1-
acid glycoprotein
Plasma protein binding
• Soluble substances
(Ions and small molecules).
They bind to plasma proteins (albumin), transferrin for iron,
ceruloplasmin for copper
• Liposoluble substances or apolar
They bind to lipoproteins (globulins: 1, 2,1, 2, ).

• Substances consigned in
R d Bl
Red Blood
dCCels
l (lead)
(l d)
Transport of Pb
in erythrocytes
LECTURE 3. Distribution

Plasma protein binding:

R
Reversible 
Reversible binding
ibl  bi
 binding
bi di
• Reduce the concentration of a unbound states in plasma
(unbound fraction exhibits pharmacologic effects,
effects it may be
metabolized and/or excreted)
•bound state…….  no activity.

The xenobiotic‐binding protein acts:
 Tampon 
p
 Reservoir of toxic substances from which the
drug is slowly released

Xenobiotic binding with protein through stable and reversible bond by


NH2 groups and-
and-SH: hydrogen bonding, ion / dipole Van der Waals
forces.
LECTURE 3. Distribution
4)Storage of chemicals in tissues

The retention of xenobiotics in tissues involves the impairment of its


main action

Characterisitcs for DISTRIBUTION AND FIXING

a) Tissue protein affinity.


In tissues, the toxin binds to the same type of
proteins in plasma (balance: free toxic <---
---> bounded Toxic)

LECTURE 3. Distribution

4)Storage of chemicals in tissues

b)) Lipid
p ((Partition coefficient))
Hg: - inorganic derivatives: in kidney
- Organic derivatives: brain and liver
organochlorine Compounds: Fat tissue

The water-soluble toxics are distributed more or less evenly throughout the
body, and rapidly eliminated.
Liposoluble toxic tends to accumulate in various tissues such as the liver,
liver
adrenal glands, adipose tissue, nerve tissue, etc.

c) Fixation
Fixation: selective chemical affinity
Fluor and lead: accumulate in
bones and interfere with the
metabolism of Ca +2

As accumulates in nails and hair interferes with sulfur amino acids

the paraquat in lungs

LECTURE 3. Distribution
3. Elimination

Elimination ((= excretion + biotransformations))

3. Excretion

Excretion takes place by:


Urine, bile, feces and exhaled air.

To a lesser extent
extent::
Lung, breast milk, placenta, sweat, saliva, tears, hair, nails,
skin, etc..
LECTURE 3. Excretion

3.1. Renal excretion
RENAL FILTER:
 Blood Flow 1000-1250 mL / min (25% in cardiac output)
y
Ie 1800 L blood / day.
 First filtration (primary urine): 130 mL / min = 190 L / day
 1% is excreted primary urine: 1
1.5
5 L / day

Hydrosoluble: No protein‐bound 
and low PM.
SUBSTANCES
Liposolubles: biotransformations for 
increasing polarity
3.1. Renal excretion
A) Glomerular Filtration
B) active tubular Secretion
C) Tubular Resorption

nephron

3.1. Renal excretion
A) glomerular filtration:
Pore glomerular capillaries 70 nm 
Pore  glomerular capillaries 70 nm 
FILTER: Water and solutes.
B) t b l  
B) tubular secretion: 
tio  
Acids, basis and metals by active 
transport (T + E).
( )
There is competition for the 
transporter (T).
transporter (T)
Greater affinity for T than for 
p
plasma proteins.
p
3.1. Renal excretion
C) Reabsorption:
It depends on the pH.
 HydroSoluble substances: Urine
 LipoSoluble substances: By passive diffusion 
 Water: By passive diffusion and antidiuretic hormone (ADH)
 Glucose and amino acids: by active transport

Factors influencing the kinetics of excretion:


1. Effect of urinary pH: Urine pH: 6.5. 
pH urine Excretion Metabolization
velocity
Acid High  
substance Low  
Basic High  
substance Low  

Increases excretion:
i  i   lk li i d b   HCO3‐
W k  id urine is alkalinized by  HCO
Weak acids:
Weak Bases: urine is acidified by ClNH4
Plasma Tubule Urine Urine Tubule Plasma
wall Acid pH Basic pH wall
HXH++X- X-+H+HX

Urine pH: range day / night
22. Polar metabolites
3. Differences between species in active secretion and 
LECTURE 3. Excretion
reabsorption
3.2. Biliary excretion
Substances Conjugated compounds, polar, nonpolar, non-
ionized, cationic, anionic
Example: flunitrazepam (Ds intake 12 h again passes fewer blood)

Elimination is against a high concentration gradient with specific 
Eli i ti  i   i t   hi h  t ti   di t  ith  ifi  
transporters that can saturate
Enterohepatic circulation:
Liver
Many organic compounds are 
combined  with glucuronide or 
sulfate ions prior to being excreted 
in the bile and excreted feces. Transport to
The intestinal microflora can  the liver via
portal vein
g
hydrolyze the conjugates to be 
reabsorbed (enteroheapatic cycle)
( )

Duodenum
Biliary excretion

3.3. Other processes of excretion


PULMONARY EXCRETION

By passive diffusion.
Toxic gases and volatile: hydrocarbons
low MW (ethyl ether) alcohols, CO, CNH, etc.

EXCRETION BY BREAST GLANDS


By passive diffusion
diffusion. The milk has a more acidic than pH7 pH7.4
pH7 4 plasma so weak
bases tend to migrate into milk.
And lipo-soluble basic substances, alcohol, nicotine,
aflatoxins, POC, Pb, Cd, complexing of Ca, mercurial, Li, thiouracils, etc..

EXCRETION BY FECES
Unabsorbed Ingested substances or
compounds excreted in the bile from the intestinal blood vessels.
Passive diffusion: lipophilic compounds (POC, dioxins and PCBs).

LECTURE 3. Excretion