Jones Ofori-Amoah ( PhD MPhil MPH BSc

DipHSM DipHRM)

Department of Pharmacology and Toxicology, School of Pharmacy, UHAS.

First ask
yourself:
what is the
worst that
can happen?
Then prepare
to accept it.
Toxicology
Then proceed 1
 OBJECTIVES

• Mechanisms of cell damage and cell death

• Biological Protective mechanisms
• Effects of toxicants on the kidney and liver
• Toxic responses to the blood

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CELLULAR TOXICITY
MECHANISMS

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Mechanisms of cell damage and cell death

• Toxic compounds damage cells in several ways.

• Cellular injury leads to a complex sequence of
events

Eventual response may be:

• reversible injury
• irreversible, leading to the death of the cell
(necrosis)

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Cell damage/death:
The process leading to cell death can be divided
into 3 events:

Primary: those that result in initial damage

Secondary: cellular changes which follow from the
initial damage
Tertiary: these are the observable and final changes

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Primary events
• Many toxic compounds are toxic following metabolism to
reactive intermediates (metabolites)

• The reactive metabolites may then initiate one or more

primary events.

• The major primary events are:

• Lipid peroxidation
• Oxidative stress
• Covalent binding to macromolecules
• Changes in thiol status
• Enzyme inhibition
• Ischaemia
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Lipid peroxidation:
 Free radical formation by –
 homolytic cleavage of a covalent bond in a molecule
 addition of an electron
 abstraction of a H atom by another radical

 Have an unpaired electron on C, N, S or O, i.e. a reactive sp.

that can react with cellular components such as
polyunsaturated fatty acids found in cell membranes

 Examples: carbon tetrachloride – a hepatotoxin

destruction of B-cells by alloxan
destruction of nerve terminal by 6-hydroxydopamine

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Oxidative stress:
• Production of active oxygen sp.
 May lead to redox cycling with electrons being donated to
yield superoxide.

Changes in thiol status:

• The cell can be overwhelmed by excessive RIs
• RIs can also oxidise GSH and thiol groups in proteins and change
the thiol status of the cell

GSH plays a key role in the protection of the cell by:

- direct chemical reaction with reactive intermediates
- a GSH transferase mediated reaction
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Enzyme inhibition:
Can be a common consequence of exposure to toxic compounds
e.g. inhibition of cytochrome aa3 by cyanide – leads to blockage of
cellular respiration

Ischaemia:
• Cessation or reduction of the supply of blood containing O2 and
nutrients such as glucose, leads to tissue damage and cell death
if prolonged
• Hypoxia and anoxia may be specific effects leading to cell death.

Covalent binding to macromolecules:

• RI can bind to macromolecules and proteins covalently.
• There is correlation between this binding and tissue damage.
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Secondary events
Changes in membrane structure and permeability – could be a
consequence of lipid peroxidation
Changes in cytoskeleton
Mitochondrial damage
Inhibition of mitochondrial function
Depletion of ATP and other co-factors
DNA damage
Lysosomal destabilization – toxic compounds may rupture lysosomes
and destroy the cell.
Changes in intracellular Ca2+ concentration ⇒⇒ leads to:
• Alterations in cytoskleton
• Activation of phospholipases which leads to breakdown of membrane
phospholipids
• Ca2+-activated proteases
• Ca2+-activated endonucleases – cleave DNA into fragments
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Tertiary Changes
• Steatosis/fatty changes
• Hydropic degeneration
• Blebbing of the plasma membrane

Morphological changes
Two distinct forms of morphological change before cell death include:
 Apoptosis: orderly process of cell shrinkage, condensation, blebbing
and phagocytosis.
 Necrosis: disorderly process involving swelling, and the rupture of
membranes, dissolution of organized structures, loss of homeostasis
and ATP.
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Apoptosis
 Occurs as part of the normal maintenance and renewal of
tissues
• Stimulated by toxic chemicals
 Triggers usually cell injury particularly involving DNA
• Cells attempt to repair injury which might fail, leading to a
mutated cell which can undergo clonal expansion.
• the cell may die by apoptosis to prevent clonal expansion of a
potentially initiated cell.
• The exposure to a sufficient dose of a chemical can initiate a
process of gene activation leading to the synthesis of specific
proteins that drive the apoptotic machinery.
• Gene products involved include fos, myc jun, bax, p53, caspases

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Necrosis
 Occurs due to irreversible damage followed by a
series of degenerative changes such as hydrolysis of
cellular components and denaturation of proteins.

 Necrosis is associated with:

• massive increase in cell volume
• swelling of the mitochondria & ER
• appearance of vacuoles and the accumulation of fluid
• ultimate rupture of the cell membrane and spillage of the
cytoplasmic contents into the tissue surroundings

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Biological Protective mechanisms
Biological systems possess a number of mechanisms
for protection against toxic foreign compounds

• Metabolic transformation to more polar

metabolites readily excreted is a method of
detoxification
• e.g. conjugation and sulphation of paracetamol

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Glutathione (GSH)
• Probably the most important protective mechanism.
• GSH is found in most cells and at a relatively high concentration
in the liver (5mM)
• It has a nucleophilic thiol group and can detoxify substances in
one of 3 ways

-conjugation catalysed by glutathione transferase

-chemical reaction with a reactive metabolite to form a
conjugate
-donation of a proton or hydrogen atom to reactive metabolites
or free radicals respectively

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Superoxide dismutase (SD)
Free radical or other reactive intermediates
may donate electrons to O2 form:

• Active O2 such as superoxide anion radical

which can cause cellular damage

SD removes superoxide to produce H2O2  catalase 

water + O2

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Vitamins and Amino acids

• Apart from GSH, lipid radicals and other radicals may

be removed by a number of endogenous compounds;
e.g. Vitamin E, Vitamin K

• Others include Cysteine, Ascorbate.

These compounds act as alternative hydrogen donors in
preference to the allylic H atoms of unsaturated lipids .

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TARGET ORGAN
TOXICITY

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Target Organ/Tissue Toxicity
Although any organ or tissue may be a target for a toxic compound,
such compounds often damage specific organs or tissues.

Reasons:
• Blood supply; e.g. kidney vrs skin
• The presence of particular enzymes or biochemical pathway
• The function or position of the organ
• The vulnerability to disruption or degree of specialization
• The ability of the organ to repair damage
• The presence of particular uptake systems
• The ability to metabolize the compound, and the balance of
toxification and detoxification
• Binding to particular macromolecules; e.g. melanin of the eye
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Toxic damage to the Liver
• One of the portals of entry to the tissues in the body
• Exposed to many potentially toxic substances by the GI tract from
the
-diet
-food additives
-contaminants
-drugs

• By virtue of its position, structure, function and biochemistry,

the liver is vulnerable to damage from drugs.

• Liver damage in man is however less common as only 9% of

adverse drug reactions affect the liver.
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The liver is a target organ for toxic substances for 4
main reasons
• The liver metabolizes many xenobiotics but metabolism
may not always result in detoxification. E.g. CCl4 & Paracetamol

• The liver also has an extensive role in intermediary metabolism

and synthesis. Interference with endogenous metabolic pathways
may lead to toxic effects.

• The liver receives 25% of the cardiac output. The blood

supply ensures that the liver is exposed to relatively high
concentrations of toxic substances absorbed from the GIT

• The secretion of bile may also be a factor. Biliary excretion

of foreign compounds leading to high concentrations especially
if saturation occurs; e.g. frusemide
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Types of liver injury
• Fatty liver (steatosis): accumulation of triglycerides in
hepatocytes
-triglyceride synthesis occurs in the liver, fatty liver – a direct
result in the reduced synthesis of apolipoprotein responsible for
transporting triglycerides out of the liver. E.g. alcohol, tetracycline

• Cholestatic damage: interference with the biliary system can

lead to bile stasis. e.g. chlorpromazine, rifampicin

• Cirrhosis: A chronic lesion resulting from repeated injury

and subsequent repair. May result from hepatocyte or cholestatic
damage; e.g CCl4 and alcohol

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Types of liver damage cont’d
Vascular lesions:
• Some substances may be directly cytotoxic to the
hepatic sinusoids and the endothelial cells, causing
damage and occlusion of the lumen.
• Blood flow in the liver is reduced leading to ischaemia
and necrosis; eg monocrotaline

Liver tumours:
Both benign and malignant tumours have been
associated with the use of oral contraceptives and
exposure to aflatoxins
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Detection of liver damage
• Bilirubin levels increase in liver damage
• Plasma albumin levels also decrease in liver damage
• Liver enzymes (such as AST, ALT, ALP) which are raised
several fold within 24 hours of liver damage, may also
be measured in plasma
• Urinary excretion of conjugated bilirubin
• Light and electron microscopy
• Liver weight/body weight ratio

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Paracetamol toxicity
• Paracetamol, widely used as an analgesic and
antipyretic
o Relatively safe in therapeutic doses
• In over-dosage, paracetamol causes hepatic
necrosis; sometimes accompanied by renal damage
and failure
• Now common for overdose of paracetamol to be
taken for suicidal intent, abortive purposes, etc.
 E.g. in the UK, around 200 deaths a year from overdose of
paracetamol

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The metabolites of paracetamol

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Proposed metabolic activation of paracetamol to a toxic, reactive
intermediate N-acetyl-p-benzoquinone imine (NAPQI). NAPQI can react
with GSH to form a conjugate or with tissue proteins.

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Treatment of paracetamol over-dosage

• N-Acetylcysteine: acts by several mechanisms

-it promotes the synthesis of GSH utilized in
the conjugation of the reactive metabolite NAPQI

-It stimulates the synthesis of GSH used in the

protection of protein thiols

-it may relieve the saturation of sulphate conjugation

which occurs during paracetamol over-dosage

- it may be directly involved in the reduction of NAPQI

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• NAC originally thought to be effective only when administered 10-12 hours
after an over-dosage.
• But now found to be effective even after 15 hours or more after over-
dosage.
• Arguably, majority of the metabolic activation and covalent binding to
protein would have taken place within 12 hours and it appears unlikely
that it will be reacting with NAPQI.

• NAC is suspected to protect liver cells against the subsequent changes

which occur after the reaction of the RI with cellular constituents.

• Methionine: not effective when given at such later times since synthesis of
GSH from methionine is inhibited or compromised by paracetamol toxicity.
• Inhibition of thiol containing enzymes involved in the synthetic pathway
likely to be the cause.

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Toxicity to the kidneys
The kidney is a target for toxic agents for the following reasons:

• Blood through the kidneys represent 25% of the Cardiac output

despite the kidneys forming 1% of body mass – High exposure to
toxins

• Concentration ability of the kidneys: many substances are

re-absorbed after glomerular filtration
• concn of toxicants in the tubular lumen can be extremely
high; e.g. sulphonamides causing crystalluria.

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Kidney as a target organ for toxicants
• Active transport: compounds which are actively transported
from the blood into the tubular fluid may accumulate in the
proximal tubular cells especially at high concns where saturation
of the transport system occurs.

• Concentration to which tubular cells are exposed may be higher

than in the blood stream. E.g. cephaloridine, which causes
proximal tubular damage.

• The kidney does not contain much cyt. P450 enzymes, but there
is sufficient to activate certain substances; e.g. Chloroform &
paracetamol.

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Detection of kidney damage
• Variety of ways of detecting kidney damage ranging from
simple qualitative tests to more complex biochemical assays

 Simple tests:
• Urine volume
• pH
• SG measurements
• Presence of cells or protein in urine
• Kidney wt/body wt ratio

• Clinically overt pathological damage detected by light or

Electron microscopy.

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 Damage can also be detected by measurement of a variety of
urinary constituents.
• Damage to tubular cells results in the leakage of enzymes such as -
glutamyltransferase & N-acetylglucosaminidase into the tubular fluid.
 Damage may also reflect in altered renal tubular function
leading to:
- the excretion of glucose and amino acids
 measurement of urea & creatinine (raised) will also indicate renal
dysfunction
 Commonest measurement is the measurement of Urea or
blood urea nitrogen (BUN)
 Clearance of the polysaccharide inulin is a better indicator
of G.F., as it is less affected by protein metabolism
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Sites for Nephrotoxicity
Difficult to define precisely the specific site of toxicity as
several tissue components may be affected:

This is because:
• Cell damage may occur due to interruption of one or
several of the many essential cellular functions rather than
from interaction with specific cellular type.

• Certain kidney cells may be particularly sensitive because

they are exposed to very high conc. of the compound
leading to a non-specific effect.
 E.g. many compounds affect the proximal tubule.
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• Proximal convoluted tubule: site of absorption of glucose and
amino acids; sensitive to metals such as chromium

• Pars recta of the proximal tubule: Main site of secretion of

organic compounds, contains the highest concn of enzymes;
sensitive to mercury, cephaloridine

• Loop of Henle: site of damage from chronic exposure to

analgesics; e.g. Aspirin, phenacetin

• Distal convoluted tubule: involved in acidification of urine;

affected by amphotericin

• Collecting ducts: Insensitive to most toxicants; site of damage

from outdated tetracyclines
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Glomerulus: Primary site of damage by several
compounds, also sensitive to immunological damage

Basement membrane: main barrier to filtrate. Toxicity

can lead to change in permeability of this membrane
causing loss of proteins; damaged by puromycin

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Toxicity to kidneys:
 Aminoglycosides
 Important cause of renal damage in humans
• They are carbohydrates with glycosidic linkages to side chains
containing various amino acids.
 E.g. Gentamycin has 5 amino groups – cationic (achieves a concn
in the renal cortex 2-5 times more than in the blood).
• There appears to be a correlation between the number of
ionisable groups & nephrotoxicity
• It is believed that the cationic aminoglycosides bind to anionic
phospholipids of the proximal tubule.
 Bound aminoglycoside is engulfed by endocytosis and stored in
lysosomes
 Lysosomes eventually rupture and release hydrolytic enzymes to
mediate the cellular damage. 37
 Cephalosporins
E.g. Cephaloridine causes kidney damage in humans
 Acute doses cause proximal tubular necrosis, it accumulates
in the cortex.
• An organic anion, but has a cationic pyridyl group and is
actively secreted by the cationic system into the tubular
lumen.
• There is also an anionic transport system for re-uptake into
the tubular cell which is more active
 the overall effect is accumulation
• Movement of cephaloridine via facilitated diffusion out of the
tubular cell into the lumen also seems to be restricted hence
intracellular concn remains high.
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Blood as a Target Tissue
 Almost all foreign compounds are distributed via the
bloodstream
• Components of blood are exposed at least initially to high
concns of foreign compounds

 Damage to and destruction of blood cells result in a variety of

events leading to reduced ability to carry oxygen to the tissues
if red cells are involved. E.g. Aniline, dapsone, nitrobenzene
 Their reactive intermediates (hydroxylamines) are unstable and can be
further oxidized to reactive products in the presence of oxygen in the
red cell and damage Hb.

 Hb may then be oxidized to methHb and be unable to carry O2

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• Irreversible damage to Hb can occur as a result of oxidation of
thiol groups in the protein.

• Proteins become denaturated leading to haemolysis of red

cells. E.g. Phenylhydrazine

• The bone marrow is a target for toxins as the cells are rapidly
dividing;
 e.g. Benzene, chloramphenicol will attack dividing cells in
the bone marrow to induce aplastic anaemia.

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