Liver nicnas-nov-2012

An Introduction to the Toxicology of the Liver
& Rodent Stomach.
Rhian B. Cope BVSc BSc(Hon 1) PhD DABT ERT

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Yes, there is a lot of basic science.

It is included deliberately: if you do not understand
the fundamentals of how and why the liver reacts to
xenobiotics, you cannot really understand the
significance and human-relevance of the changes
that occur.

Understanding the mode of action is the key to just
about everything in toxicology and toxicological risk
assessment.

Please bear with me.

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Sections.
Section 1: A Revision of the Basic Anatomy and Physiology of the Liver,
Reasons for the Susceptibility of the Liver to Toxic Injury and Classical
Clinical Signs of Hepatic Disease.

Section 2: Responses of the Liver to Toxic Injury

Section 3: Interpretation of Rodent Hepatic Tumour Data: The Human-
Relevance Framework

Section 4: Detection/ Measurement/Assessment of Hepatic Toxicity.

Section 5: The Two Basic Classes of Hepatic Toxicants, and Classical “Must
Know” Agents Causing Hepatic Damage.

Section 6: Interpretation of Rodent Stomach Tumour Data: The Human-
Relevance Framework.

Section 7: Case Studies.

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Section 1.

A Revision of the Basic Anatomy and Physiology of the
Liver, Reasons for the Susceptibility of the Liver to Toxic
Injury and Classical Clinical Signs of Hepatic Disease.

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Learning Tasks Section 1.
1. Describe and understand the toxicologically significant features of the
hepatic circulation.
2. Describe and understand the structure and toxicologically significant
features of the liver lobule.
3. Describe and understand the structure and toxicologically significant
features of the liver acinus.
4. Understand the toxicological significance of Kupffer, Pit and Ito cells.
5. Describe and understand the key physiological roles of the liver and the
potential effects of disrupting these functions.
6. Describe and understand the toxicologically significant features of bile
formation/excretion and excretion of bilirubin.
7. Describe and understand the basis for the susceptibility of the liver as a
toxic target organ.
8. Describe and understand the classical clinical signs of hepatic disease.

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Hepatic Circulation and Blood Supply.

•Key points:
Liver receives blood via two routes:
high oxygen blood from the hepatic
artery (30%) and low oxygen blood
from the portal vein (70%).
Blood leaves the liver only by the
hepatic vein.
Liver is placed between venous
blood returning from the bulk of the
GI and peritoneal cavity and the
venous arm of the systemic
circulation.
WHAT ARE THE TOXICOLOGICAL


CONSEQUENCES OF THIS?

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Structure of the Liver Lobule.

Low magnification view ofBthe a liver lobule in the pig
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01/05/07 Low magnification view B Cope human liver lobule
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Note the lack of an
endothelial basement
membrane, large
endothelial pores and
large endocytic vacuoles.
What are the key
toxicological
consequences of these
features?

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Structure of the Liver Acinus.

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• Acinar zone 1 approximates “Periportal” using the
“Lobular” system.

• Acinar zone 3 approximates “Centrilobular” using the
“Lobular” system.

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Describe the distribution of damage (necrosis) in this liver
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section using the “lobular” and “acinar” system.

?

Describe the distributionDr R B Cope
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of damage (necrosis) in this liver
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Central Vein

Describe the distributionDr R B Cope
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of damage (necrosis) in this liver
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Central Vein

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Centrilobular orB Zone 3 Necrosis.
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• Hepatocytes are generated in zone 1 from their primordial stem
cell and migrate from zone 1 to zone 3 before undergoing
senescence/apoptosis in zone 3.

– The youngest hepatocytes occur in zone 1, the oldest occur
in zone 3.

– The hepatocyte cycle in the rat is approximately 200 days.

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• All hepatocytes are NOT equal. Important
functional/physiological differences occur between
hepatocytes in different acinar zones.

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Hepatocyte Zonal Specialization.
Parameter Zone 1 Zone 2 Zone 3
Oxygen tension and level High Intermediate Low
of nutrients in blood supply
Exposure to portal blood First site of Intermediate Last site of exposure
exposure
Glutathione levels High Intermediate Low
Bile acid excretion High Intermediate Low
Overall balance between Relatively Intermediate Phase I predominates
Phase I and Phase II balanced over Phase II
metabolism
CYP level (particularly Cyp Lower Intermediate High
2E1)
Level of fatty acid High Intermediate Low
oxidation, gluconeogeneis,
and ureagenesis
Concentration of materials High Intermediate Low
(bile salts, bilirubin,
excreted compounds) in
adjacent bile canaliculus

Number of mitochondria High Intermediate Low
Glycogen and other High Intermediate Low
nutrient stores

Toxicological Consequences of Hepatocyte Zonal Specialization.
Parameter Zone 1 Zone 2 Zone 3
Oxygen tension and level
of nutrients in blood supply
Exposure to portal blood
Glutathione levels
Bile acid excretion
Overall balance between
Phase I and Phase II
metabolism
CYP level (particularly Cyp
2E1)
Level of fatty acid
oxidation, gluconeogeneis,
and ureagenesis
Concentration of materials
(bile salts, bilirubin,
excreted compounds) in
adjacent bile canaliculus

Number of mitochondria
Glycogen and other
nutrient stores

Kupffer Cells.
• Kupffer cells are the resident tissue macrophage of the
liver. Located in the sinusoids.

• Large number of Kupffer cells are present in the liver:
80% of body‟s resident tissue macrophages.

• Fully functional macrophage: can trigger inflammation
and act as antigen presenting cells.

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Kupffer Cells.
• Of considerable importance in hepatic toxicology:

– Activation during inflammation results in the
generation of various free radicals e.g. superoxide
anion, peroxynitrite, nitrogen oxides

– Triggering and participation in inflammation.

– Accumulation of iron (hemosiderin, ferritin).

– Degradation of heme.

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Pigment accumulation within Kupffer cells.
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Pit Cells.
• Located in the space of Disse.

• Function as NK or LAK cells.

• Important in inflammation.

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Ito Cells.
• Synonyms = “fat cells”, stellate cells.

– Two major roles:

• Storage of Vitamin A.

• During inflammation or liver damage, produce
collagen i.e. responsible for hepatic fibrosis.

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Congestive cirrhosis (replacement of hepatocytes with fibrous tissue)
secondary to right sided heart failure, trichrome stain. Remember: Ito cells
are responsible for the laying down of new collagen within the liver. WHAT
ARE THE CRITICAL FUNCTIONAL CONSEQUENCES OF SUCH A
REACTION IN THE LIVER?

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A Concise Summary of Key Hepatic Functions

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Consequences of Disruption of Hepatic Function
Consequences

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Bile Formation and Hepatic Excretion.
• Bile formation involves both hepatocytes and
cholangiocytes

• Bile formation involves 8 basic processes:

1. Materials that undergo biliary excretion move from the sinusoid
through the space of Disse and through the basolateral
hepatocyte cell membrane via diffusion, active transport or
endocytosis.

2. The materials for excretion are transported across the
hepatocyte with or without metabolism and storage and then
actively transported into the canaliculi.

3. Vesiclular transport involves the detachment of lipid vesicles
from the apical hepatocyte membrane to form bile micelles.
Bile micelles contain lipophilic compounds, bile salts,
cholesterol, phospholipids, and high molecular compounds

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4. Excretion of compounds is sufficient to generate osmotic water
flow into the bile canaliculi.

5. Forward movement of bile within the canaliculi occurs by ATP-
dependent peristaltic contraction of the actin-myosin web
located underneath the apical membrane of the hepatocytes.

6. Within the bile ductules and common hepatic duct, bile
composition and volume are modified by cholangiocytes:

7. Volume increases due to the osmotic gradient created by the
active excretion of HCO3- in exchange for Cl- by cholangiocytes;
~ 40% of bile volume is due to this excretion mechanism.

8. Cholangiocyte re-uptake of some constituents (some bile acids)
occurs.

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• Molecules with a molecular weight of ≤ 300 Da are
more efficiently excreted in bile than molecules with a
greater molecular weight.

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Major Hepatocyte and Cholangiocyte
Transporters involved in Bile Formation

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Major Hepatocyte Involved in Bile Formation
Basolateral Transporters Function

Na+-taurocholate-co-transporting Uptake of conjugated bile acids,
peptide (NTCP) estrogens

Organic anion transporter Uptake of amphiphilic compounds,
polypeptide (OATP) steroid conjugates, neutral steroids,
sulfobromophthalein (OATP2),
bilirubin (OATP2), glutathione
conjugates, leukotriene s, C4
organic cations, small peptides,
digoxin

Organic cation transporter I (OCT I) Divalent lipophilic cations,
xenobiotics that contain a tertiary or
quarternary amine group
Bilitranslocase Bilirubin, sulfobromophthalein;
inhibited by phenylmethyl-sulphonyl
fluoride; exists in two metastable
forms: high and low affinity.

Major Hepatocyte Transporters Involved in Bile Formation.
BasolateralTransporters Function
Organic anion transporter 2 (OAT2) Uptake of indocyanine green, and
nonsteroidal anti-inflammatory
drugs, such as ketoprofen,
indomethacin, and salicylates
through the basolateral hepatocyte
cell
membrane

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Major Hepatocyte Transporters Involved in Bile Formation.
Apical Transporters Function
Multidrug resistance proteins Excretion of cationic and lipophilic
(MDR), particularly MDR1 compounds. MDR1 has no physiological
substrate in non-ruminants; function is
(Note: MDR1 = p-glycoprotein, the secretion of amphiphilic cationic
which has now been renamed the xenobiotics, steroid hormones,
ATP-binding cassette sub-family B hydrophobic pesticides and glycolipids;
member 1 transporter, or ABCB1) responsible for phyloerythrin excretion
in ruminants!

SPGP = bile salt export pump Transports monoanionic bile salts.
(BSEP)
Multidrug resistance-associated Excretion of glucuronic acid, sulfate
proteins (MRP); MRP2 = canalicular and glutathione (anionic)
multispecific organic anion conjugates, phospholipids;
transporter (cMOAT) Excretion of mono- and diglucuronic
acid bilirubin conjugates (MRP2)
and glutathione-
sulfobromophthalein conjugates
(MRP2)

Hepatic Bilirubin Excretion.
Heme containing proteins (Hb,
Mb, CYP450) Hepatocyte

Sinusoid
Reticuloendothelial system
Alb

Bile canaliculus
Spleen, Kupffer cells,
Free heme (red)

UDP-glucuronide
Heme
OATP *
oxygenase
Br Br
Biliverdin (green)
Biliverdin
Alb-Br
BT *
reductase

UGT-1A1
Bilirubin (Br;brown) MRP2 *
Albumen

Space of Disse
(ALB)

Conjugated Br
Alb-Br Systemic Gluc-Br
(Gluc-Br) in Bile
Circulation
(“Free” or unconjugated Br)

*Organic anion transport protein; *Bilitranslocase; * Rate limiting step for bilirubin excretion

Extrahepatic Aspects of Bilirubin Excretion.

• Conjugated bilirubin excreted in the bile is converted by
bacterial action within the ileum and colon into
urobilinogen which undergoes enterohepatic circulation.

• Urobilinogen that is not taken up and re-excreted by the
liver passes into the systemic circulation and is excreted
by the glomerular filtration in the kidneys

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Extrahepatic Aspects of Bilirubin Excretion.

• The amount of urobilinogen formed, and thus excreted
by the kidneys increases dramatically with increased
formation of bilirubin (e.g. hemolysis).

• The amount of urobilinogen in urine will decrease with:
– Severe cholestasis (failure of conjugated bilirubin
excretion).
– Bile duct obstruction.
– Severe disruption of the GI microflora (antibiotics).

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Important Aspects of Bilirubin Excretion.
• The excretion of conjugated bilirubin is inhibited by the
administration of sulfobromophthalein due to competition for
the MRP2 transporter.

• Impaired hepatic sulfobromophthalein excretion (i.e.
increased or delayed retention) has at least three potential
causes:

– Cholestasis due to impaired apical excretion.
– Inhibition of glutathione-S-transferases (requires
conjugation to glutathione for excretion).
– Impaired basloateral bilitranslocase and OATP function.

* note: bromosulfonphthalein (BSP) was a commercial brand name for
sulfobromophthalein. Older literature will often refer to a BSP test
which simply means a test for plasma clearance of
sulfobromophthalein.
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• Bilirubin in plasma is measured by the van den Bergh
assay which makes two different measurements: total
bilirubin and direct bilirubin.

• Classically, the direct bilirubin is regarded as a measure
of conjugated bilirubin in plasma.

• Indirect bilirubin (unconjugated) = total bilirubin – direct
bilirubin.

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• Modern analytical methods have now demonstrated that
plasma from normal individuals contains virtually no
conjugated (i.e. “direct”) bilirubin.

• Elevations of plasma direct or conjugated bilirubin
primarily occur with:
– Obstruction of the bile ducts or canaliculi.
– Decreased canalicular contraction.
– Inhibition of MRP2.
– Hepatocellular disease.

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Bilirubin Excretion in the Neonate.
• Bilirubin excretion, like most hepatic excretion, takes
time to develop in neonates.

• Bilirubin produced by the fetus is cleared by the placenta
and eliminated by the maternal liver.

• After birth, the neonatal liver slowly develops the
capacity for bilirubin clearance and excretion.

• Levels of UGT1A1 in neonatal hepatocytes are low and
unconjugated bilirubin is excreted into the gut.

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• The neonatal gut lacks the microflora to convert bilirubin
to urobilinogen and bilirubin undergoes enterohepatic
cycling.

• Levels of MRP2 are also low in the neonate. Remember
transport of conjugated bilirubin across the hepatocyte
apical cell membrane is the rate-limiting step for bilirubin
excretion.

• Neonates typically have elevated free bilirubin in their
plasma due to impaired excretion by MRP2 and
enterohepatic cycling.

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• Any xenobiotic that increases the production of
bilirubin in the neonate will produce rapid, large
increases in plasma bilirubin.
– Any agent that produces hemolysis or defective
erythrogenesis.
– Any agent that produces hemorrhage.
– Any agent that produces cholestasis.

• This results in a condition called kernicterus
(bilirubin encephalopathy) in which bilirubin crosses
the blood-brain barrier and precipitates within the
basal ganglia and other sites in the brain resulting in
CNS damage. Yellow staining of brain nuclei due to
bilirubin precipitates is the classical pathology
associated with kernicterus.

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Globus pallidus staining with bilirubin
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Basis for the Susceptibility of the Liver to Toxicity.

• Position within the circulatory system: high exposure to
xenobiotics absorbed via the GI (also peritoneum) i.e.
first pass effect.

• High level of biotransformation, and therefore, significant
risk of generating reactive metabolites.

• Susceptibility to oxidant injury.

• Susceptibility to hypoxic injury (centrilobular).

• Critical biosynthetic/homeostatic functions.

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• Ability to concentrate xenobiotics within the biliary tree,

• Large tissue macrophage population: inflammation and
oxidative injury.

• Little or no selectivity of sinusoidal endothelium (large
pores).

• Capacity to separate xenbiotics from albumen and other
carrier proteins.

• Capacity to accumulate metals, vitamin A and other
xenobiotics.
• Liver has high energy consumption and Is susceptible to
agents that affect mitochondrial function.
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• Enterohepatic circulation can result in sustained exposure to
xenobiotics.

• Lipophilic xenobiotics tend to concentrate within the liver
since it is relatively rich in cell membranes

• Substrates for the transporter systems of the basolateral
hepatocyte membrane also tend to selectively accumulate in
the liver e.g. phalloidin, microcystin.

• Compounds that have hepatic storage can cause toxicity e.g.
iron (stored as ferritin), cadmium (stored as a Cd-
metallothionine complex), vitamin A (selectively stored in Ito
cells)
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Patients showing clear evidence of
jaundice: yellow discoloration of the skin
and sclera.
Important differential is high dietary beta
carotene – tissues and skin are stained
yellow, but the sclera remains white!

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Clinical Signs of Acute Hepatocellular Disease.

• Markers of malaise i.e. fatigue, weakness, nausea, poor
appetite.

• Icterus/jaundice: probably the best clinical marker of
severity. Indicates bilirubin level > 2.5 mg/dl.

• Spider angiomata and palmar erythema.

• Itching (self mutilation in animals).

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Clinical Signs of Acute Hepatocellular Disease.

• Right upper quadrant abdominal pain.

• Abdominal distention.

• Intestinal bleeding.

• ± Heatomegaly.

• Bilirubinuria: dark characteristically colored urine

• In many cases of hepatocellular disease, there are no
clinical signs. Cases are recognized by biochemical liver
tests.

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Clinical Signs of Advanced or Chronic
Hepatocellular Disease.

• Weight loss, muscle wasting.

• Evidence of hemorrhage and coagulopathy.

Evidence of
• Ascites. inadequate serum
protein synthesis.
• Edema of the extremities.

• Fetor hepaticus = typical sweet ammoniacal odour of
patients with hepatic failure (failure of ammonia
clearance/metabolism).

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Ascites following severe liver disease. Note the eversion of
the umbilicus.
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Mid-level abdominal CT scans. Left = normal; Right = ascites
secondary to liver failure.

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• Hepatic encephalopathy (change in sleep patterns,
change in personality, irritability, mental dullness,
disorientation, stupor, asterixis*, flapping tremors of body
and tongue, coma).

• Caput medusa = development of prominent collateral
veins radiating from the umbilicus due to the
recanulation of the umbilical vein and its tributaries due
to portal hypertension and porto-systemic shunting.

* Asterixis = a motor disturbance marked by intermittent lapse of an assumed
posture due to intermittent sustained contraction of muscle groups;
characteristic of hepatic coma; often assessed by asking the patient to write or
draw simple pictures (e.g. draw a clock face).
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Caput medusae associated with portal hypertension,
portosystemic shunting and severe liver disease.
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• Hepatorenal syndrome: characterized by progressive
renal failure that develops following chronic liver disease
+ ascites and other evidence of liver failure. Mechanism
is unknown but the syndrome is associated with altered
renal hemodynamics and altered prostaglandin levels
are implicated.

• Portal hypertension, portosystemic shunting and acute
venous hemorrhage due to rupture of abdominal veins.

• Spontaneous bacterial peritonitis (failure of bacterial
opsonization due to low albumen and other opsonizers).

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• Hepatopulmonary syndrome: development of right to left
intrapulmonary shunts in advanced liver disease.
Mechanism is unknown but involves altered pulmonary
nitric oxide levels.

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Clinical Signs of Advanced Hepatocellular or Cholestatic
Disease in Ruminants: Secondary Photosensization.
In ruminants:

Rumen bacteria
Chlorophyll Phylloerythrin

Absorbed

Hepatocyte

Transported across the apical
Excreted in bile hepatocyte cell membrane by ATP-
binding cassette transporter B1 [p-
glycoprotein or MDR 1)

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Clinical Signs of Advanced Hepatocellular or Cholestatic
Disease in Ruminants: Secondary Photosensitization.

• Prolonged inhibition of ABCB1, cholestasis or
hepatocelular disease in ruminants results in an
accumulation of phylloerythrin within the circulation and
tissues.

• Phylloerythrin absorbs light and acts as a photosensitizer
within the skin resulting in severe skin inflammation and
sloughing.

• Disease in sheep (particularly associated with
sporodesmin-induced liver disease) is colloquially called
“facial eczema.”

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Secondary photosensitization of the face due to
01/05/07 sporodesmin poisoning in a sheep
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Severe secondary photosensitzation of the udder of a cow
with advanced hepatic disease (again due to sporodesmin)
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Section 2:

Responses of the Liver to Toxic Injury.

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1. Describe and understand the stereotypical cellular
responses of the liver to xenobiotic injury.

2. Describe and understand the processes involved in the
development of cholestasis.

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Stereotypical Responses of the Liver to
Toxicant Injury.
• The patterns of the hepatocellular response to toxicant injury
are generally stereotypical and not toxicant specific (although
there are exceptions to this rule).

• The hallmark of the liver’s response to toxicant injury is its
large functional reserve and large capacity for healing,
often with no significant sequelae!
– For example, a 2/3 hepatectomy is survivable and both
normal liver function and size will be restored within weeks!

– This will occur provided significant fibrosis or massive
necrosis of the lobules does not occur and the source of
injury is removed i.e. exposure is not chronic.

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Hepatocellular Adaptive Responses.

• These changes are generally reversible once
xenobiotic exposure stops.

• In terms of a toxicology study, ideally this propensity
for reversal should be tested by the inclusion of an
adequate post-exposure recovery period in the study.

• This inevitably involves inclusion of additional
experimental groups i.e. groups that is euthanitized at
the end of exposure (necropsy + histology) plus groups
that are euthanitized 14 to 30 days post exposure +
appropriate control groups.

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• Sadly this is rarely done despite the provision for this in
the OECD guidelines.

• My personal view is that histological discrimination of
the types of lesion present is not sufficient to claim
reversibility; must have actual documented study
evidence of the reversibility of hepatic adaptive
responses!

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• Represent adaptive responses to xenobiotic response
rather than hepatocellular damage per se.

• Used as histological markers of xenobiotic exposure.

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• Do not result in disease per se but are often of
significance for the toxicokinetics/toxicodynamics of
drugs and other xenobiotics and thus may significantly
influence the toxicity of particular toxins/toxicants.

• Usually detected histologically but may be visible
grossly as hepatomegaly and/or increased liver weight.

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Hepatocellular Adaptive Responses:
Centrilobular Hepatocellular Hypertrophy.

• Due to ↑ smooth endoplasmic reticulum content in
centrilobular/Zone 3 hepatocytes.

• Associated with chemical induction of CYP, particularly
CYP2E1.

• Associated with massive increases in the amount of
smooth endoplasmic reticulum.

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Centrilobular Hepatocellular Hypertrophy.

• Reversible following removal of the initiating agent.

• Example initiating agents: phenobarbital and other
oxybarbiturates, Ah receptor agonists (TCDD, PCDFs).

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Centrilobular hepatocyte hypertrophy in a mouse treated
with phenobarbital for 8 months.
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Centrilobular hepatocyte hypertrophy in a mouse treated with
phenobarbital for 8 months Note the eosinophilic cytoplasm due to
the large increase in smooth endoplasmic reticulum as a result of
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CYP (particularly CYP2E1) induction. 79

Eosinophilic Centrilobular Hepatocellular Hypertrophy.
• Due to ↑ peroxisomes in centrilobular hepatocytes.

• Prolonged eosinophilic centrilobular hypertrophy is
associated with pericanalicular lipofuscin pigment
deposition.

• Prolonged exposure to chemicals that induce
peroxisome induction may result in hepatocellular
neoplasia in rodents.

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Eosinophilic Centrilobular Hepatocellular Hypertrophy.

• Classical agents: phthalate plasticizers.

• Rodent-specific response.

• Relevance to humans is controversial!

• Currently regarded as not relevant to humans in many
jurisdictions, however this is an area of considerable
scientific challenge

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Centrilobular eosinophilic hepatocyte hypertrophy (left) in a mouse due to chronic
exposure to phthalates. Right image shows immunohistochemical staining for
peroxisomes. Note that chronic exposure to peroxisome proliferators is
carcinogenic in rodents but not humans.
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Dr R B Cope

Xenobiotic-Induced Hepatocyte Hyperplasia.

• Usually accompanied by CYP induction,
hepatomegaly, and hepatocyte hypertrophy.

• Never continues for more than a few days.

Reversion is associated with ↑ hepatocyte apoptosis.

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Derived from the UK PSD guideline
(included as an appendix to the
notes)

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Early Markers of Hepatocellular Damage:
Hepatocyte Nucleolar Lesions.

• Due to changes in RNA synthesis.

• Changes include: ↓ size, ↑ size, nucleolar
fragmentation, nucleolar segregation.

• ↓ Nucleolar size is usually an acute lesion that
occurs within hours of hepatotoxin exposure;
often the first identifiable toxic hepatic lesion.

• ↑ Nucleolar size is commonly associated with hepatic
neoplasia.

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Early Markers of Hepatocellular Damage:
Hepatocyte Polysome Breakdown.

• In normal protein synthesis, ribosomes are evenly
spaced along single strands of mRNA forming a
structure called a polysome.

• ↓ RNA synthesis  ↓ polysomes  loss of basophilic
granules in hepatocyte cytoplasm.

• Loss of basophilic granules in hepatocyte
cytoplasm implies ↓ cellular protein synthesis and
is an early marker of hepatocellular injury.

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Reversible Hepatocellular Injury:
Hydropic Degeneration.

• Accumulation of water in the cytosol or rough
endoplasmic reticulum.

• Characterized histologically by pale-staining
cytoplasm, narrowing of the sinusoids and space of
Dissė.

• Typically reversible.

• Due to failure of hepatocytes to maintain intracellular
Na+ balance.

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Hepatocyte hydropic degeneration.

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Hepatic Lipidosis (“Fatty Liver”).

• Two basic forms: Accumulation of triglycerides or
accumulation of phospholipids.

• Responses are non-specific: many other
conditions cause fatty liver and it is NOT
pathognomonic for hepatotoxicity.

• Accumulation of triglycerides within membrane-bound
vesicles in hepatocytes

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• Occurs due to an imbalance in the uptake of fatty acids
and their excretion as very low density lipoproteins
(VLDL) due either to impaired VLDL synthesis or
secretion.

• Typically associated with acute exposure to many
hepatotoxins.

• Typically reversible and usually does not involve
hepatocellular death.

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Fatty liver due to triglyceride accumulation.

– Triglycerides are located within membrane-bound
cytoplasmic vesicles.

– Occurs due to an imbalance in the uptake of fatty
acids and their excretion as very low density
lipoproteins (VLDL) due either to impaired VLDL
synthesis or secretion.

– Typically associated with acute exposure to many
hepatotoxins.

– Typically reversible and usually does not involve
hepatocellular death.
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Fatty liver due to phospholipid accumulation.

– Caused by toxins that bind to phosopholipids and
block their catabolism.

– Phosopholipids accumulate in hepatocytes, Kupffer
cells and extrahepatic cells.

– Affected cells have foamy cytoplasm.

– Lesion is reversible and does not involve cell death.

01/05/07 Dr R B Cope 92

Human liver. Fatty change due to alcohol. Note the color.
Surface will feel “greasy”.
01/05/07 93

Hepatocyte fatty change due to ethanol exposure. Note: fat
droplets appear clear due to their extraction during tissue
01/05/07
processing. 94

Fine needle aspirates of hepatocytes. Normal on
01/05/07 the left, fatty change on the right. 95

Hepatocellular Death:
Hepatocellular Apoptosis and/or Necrosis.
• Both apoptosis and necrosis occur and these
endpoints can often be regarded as points on a dose
response curve i.e. apoptosis for low exposures,
necrosis for high exposures.

• Toxins are generally specific for a single area or zone
within the hepatic lobule, although this pattern can
be altered by dose and duration of exposure.

• The significance of necrosis as an endpoint in the
liver is that it almost always occurs with
inflammation which tends to amplify the amount of
damage that occurs.

01/05/07 Dr R B Cope 96

Centrilobular, Zone 3 or Periacinar Necrosis.
• Most common reaction to toxic injury.

• Lesion is usually uniformly distributed within the liver.

• Typically, cellular injury is typically limited to
hepatocytes but destruction of the endothelium and
centrilobular hemorrhage may also occur.

• Generally rapidly repaired with minimal fibrosis in the
area surrounding the central vein.

01/05/07 Dr R B Cope 97


• Centrilobular necrosis can be triggered by ↓ blood flow
since this is the area of the lobule that receives blood
last, is the most hypoxic and is the most nutrient-
limited.

01/05/07 Dr R B Cope 98

• Metabolic basis for the pattern (i.e. metabolic zonation)
is that the centrilobular hepatocytes have the highest
levels of CYP and therefore the highest activation of
xenobiotics to potentially toxic metabolites.
•
– In this area, phase I and phase II metabolism are out of
balance.

– Phase I metabolism often converts xenobiotics to electrophilic
metabolites. Phase II metabolites are usually stable and non-
reactive.

– If phase I predominates over phase II metabolism, the
tendency for production/accumulation of reactive electrophilic
metabolites is higher, thus there is a greater tendency for
01/05/07 hepatocellular injury. 99

Centrilobular necrosis.
01/05/07 Dr R B Cope 100

Hepatic centrilobular necrosis.
01/05/07 Dr R B Cope 101

Periportal or Zone 1 Necrosis.
• Less common than centrilobular necrosis.

• Hemorrhage is rarely associated with periportal
necrosis.

• Inflammatory response is usually very limited or
absent.

• Repair is usually rapid with minimal fibrosis.

• Repair is often accompanied by bile ductule
proliferation which usually regresses over time.

01/05/07 Dr R B Cope 102

Periportal or Zone 1 Necrosis.

• Pathophysiological basis for periportal necrosis.

• Periportal area receives blood first and is thus the
first area to be exposed to xenobiotics and is also
exposed to the highest concentration of xenobiotics.

• Metabolic zonation effects: area has the highest
oxygen tension.

01/05/07 Dr R B Cope 103

Periportal degeneration and portal cirrhosis.
01/05/07 Dr R B Cope 104

Massive or Panacinar Necrosis.
• Massive wide-spread death of hepatocytes with only a
few or no survivors.

• Involves the whole lobule; not all lobules are equally
affected.

• Necrosis extends from the central vein to the portal
area (bridging necrosis).

01/05/07 Dr R B Cope 105

Massive or Panacinar Necrosis.
• Severe panacinar necrosis and destruction of the
supporting structures usually results in ineffective
repair i.e. variably sized regenerative nodules that lack
normal lobar structure; significant permanent fibrosis
usually occurs.

• Usually occurs following exposure to massive doses of
hepatotoxins or when toxins are directly injected into
the portal venous system.

• In the case of intravascular injection of the toxin,
massive necrosis may be confined to specific liver
lobes due to incomplete mixing of the agent in the
portal vascular supply.

01/05/07 Dr R B Cope 106

Hepatic massive necrosis. Note the periportal
accumulation of bile pigments.
01/05/07 Dr R B Cope 107

Hepatic massive necrosis.
01/05/07 Dr R B Cope 108

Cirrhosis.
• Cirrhosis = hepatic fibrosis + nodular regeneration.

• 2 basic forms:
– Centrilobular (i.e. inside  outside fibrosis).
Usually occurs secondary to chronic right sided
heart failure and/or hepatic vein hypertension.

– Periportal (i.e. outside  inside fibrosis). Usually
occurs secondary to repeated episodes of
hepatocellular necrosis or following an episode of
massive necrosis or chronic/significant damage to
the sinusoidal vasculature.

01/05/07 Dr R B Cope 109

Nodular regeneration and periportal cirrhosis following massive necrosis. Trichrome
stain. Note that the regenerating liver nodules vary in size and are highly
disorganized. There is no regular lobular structure and extensive periportal fibrosis
is present. What do you think the functional consequences this lesion are?

01/05/07 Dr R B Cope 110

Cirrhosis.
• Regenerating hepatocyte lobules nodules do not have
the normal lobular structure and vary in size.
Inevitably hepatic function is significantly
compromised.

• Irreversible, usually progressive and typically has a
poor prognosis.

01/05/07 Dr R B Cope 111

Hepatocyte Megalocytosis.
• Characterized by the appearance of large
multinucleate hepatocytes in areas of hepatocellular
regeneration.

• Megalocytes are hepatocytes that have undergone cell
division but cannot complete cell separation.

• Sign of frustrated or ineffective hepatocyte proliferation
i.e. suggests a blockage in the cell division process.

• Classically associated with the pyrrolizidine alkaloids,
but also occur with several hepatic carcinogens.

01/05/07 Dr R B Cope 112

Bile Duct Hyperplasia.
• Common response to xenobiotics.

• May be restricted to the periportal area or may extend
beyond the periportal area.

• Simple bile duct hyperplasia is not associated with
cholangiofibrosis.
– May remain static, regress or progress.

01/05/07 Dr R B Cope 113

Bile Duct Hyperplasia.

• Cholangiofibrosis.
– Characterized by proliferation of bile ducts
surrounded by fibrous tissue.

– May regress over time following removal of the
initiating agent but is generally regarded as a more
serious type of injury due to the fibrosis.

01/05/07 Dr R B Cope 114

Periportal Bile duct hyperplasia.

01/05/07 Dr R B Cope 115

Hepatocellular Death: Focal Necrosis.

• Randomly distributed death of single or small clusters
of hepatocytes.

• Uncommon.

• Usually accompanied by mononuclear cell infiltration at
the lesion site.

• Pathophysiological basis for the lesion is poorly
understood.

01/05/07 Dr R B Cope 116

Damage to the Sinusoidal Epithelium:
Peliosis Hepatis and Related Syndromes.
• Progressive damage to the sinusoidal endothelium
results in eythrocyte adhesion, eventual blockage of
the sinusoidal lumen and hepatic engorgement.

• Typically associated with pyrrolizidine alkaloids.

01/05/07 Dr R B Cope 117

Damage to the Sinusoidal Epithelium:
Peliosis Hepatis and Related Syndromes.

• Peliosis hepatis: characterized by clusters of greatly
dilated sinusoids that occur randomly through the liver
parenchyma.

• Occasionally associated with other toxins that damage
the hepatic endothelium, but also occurs
spontaneously in rodents

01/05/07 Dr R B Cope 118

Lesions of Ito Cells:
Ito Cell Hyperplasia and Spongiosis Hepatis .
• Enlargement is associated with hypervitaminosis A.

• Ito cell proliferation is often associated with
centrilobular injury; under these circumstances, Ito
cells produce collagen and are responsible for inside
 outside cirrhosis.

• Spongiosis hepatis.
– Found only in rodents.
– Due to proliferation of abnormal Ito cells.
– Due to aging or exposure to hepatocarcinogens.

01/05/07 Dr R B Cope 119

Lesions of Kupffer Cells:
Iron, Endotoxin and Ricin.

• Kupffer are the primary site of iron storage in the liver
and damage occurs with iron overload.

• Kupffer cells are the primary site of uptake of
endotoxin/LPS in the liver. This may result in Kupffer
cell activation and secondary damage to hepatocytes
due to inflammation or death of the Kupffer cells.

• Kupffer cells are preferentially damaged by ricin.

01/05/07 Dr R B Cope 120

Hepatocellular Pigmentation.
• Glycogen accumulation.
– Appears as a clear cytoplasm with indistinct
vacuoles; identifiable using periodic acid-Schiff
(PAS) staining.
– Due to either up-regulation of glycogen synthesis or
impaired glycolysis.

• Lipofuscin.
– Normally accumulates with aging, but ↑ deposition
occurs following exposure to peroxisome
proliferators.
– Stains brown with H & E; special stain is Schmorl's
stain; autofluoresces under UV light.
– Lipofuscin is due to the lysosomal accumulation of
partially digested lipids.

01/05/07 Dr R B Cope 121

• Ferritin/hemosiderin.

– Excess iron is stored as ferritin (conjugate of iron +
apoferritin) or hemosiderin (incomplete breakdown
product of ferritin) in membrane bound granules
(siderosomes) particularly in Kupffer cells.

– Appears as golden brown granules in H & E
sections; special stain is Pearl‟s Prussian blue.

– Often has a pericanalicular distribution.

– Due to excessive iron intake, excessive erythrocyte
destruction or some hepatotoxins.
01/05/07 Dr R B Cope 122

• Copper.

– Appears as enlarged hyperchromatic hepatocytes +
necrosis + granulocytic/monocytic infiltrate.

– Special stains are rubeanic acid or rhodamine.

– May also be associated with Mallory body formation
(Mallory bodies are red globular accumulations in
the cytoplasm which are composed of cytoskeletal
filaments).

01/05/07 Dr R B Cope 123

Oval Cell Hyperplasia.
• Response is peculiar to rodents; Extensive oval cell
hyperplasia is only rarely observed in non-rodent
species.

• Oval cells are presumed to be hepatocyte stem cells.

• Occurs under two circumstances:

– Hepatocyte proliferation following hepatocyte
necrosis.
• Oval cells are most numerous when hepatocyte
regeneration is partially or completely blocked
e.g. with repeated insults or chronic exposure to
a toxicant.

– Exposure to hepatic carcinogens.
01/05/07 Dr R B Cope 124

Oval Cell Hyperplasia.

• Can occur independently or concurrently with bile duct
hyperplasia.

• Response is always regarded as potentially neoplastic.

01/05/07 Dr R B Cope 125

Oval cell hyperplasia in a mouse exposed to a hepatic
carcinogen.
01/05/07 Dr R B Cope 126

Hepatic Neoplasia.
• Involves hepatocellular neoplasia, bile duct neoplasia,
endothelial neoplasms and Kupffer cell neoplasms.

• Very common reaction to many carcinogens in rodent
toxicology models:
– ~ 50% of carcinogens cause hepatic neoplasia in
rodents.
– This is significantly different from humans where
hepatic neoplasia is relatively uncommon: this
remains a significant area of controversy and
concern in terms of risk analysis and regulatory
toxicology. Are agents that produce rodent liver
tumors really of great significance to humans??
(Answer: depends on the mechanism)
01/05/07 Dr R B Cope 127

Hepatic Neoplasia.
• Hepatocyte neoplasias.

• Marked strain difference in rate of spontaneous
hepatocellular carcinomas in rodents (~ 30 – 50%
incidence in C3H mice versus < 5% in male
C57B1/6 mice)
• Malignant hepatocyte neoplasias = hepatocellular
carcinomas.
• Benign hepatocyte neoplasias = hepatocellular
adenoma.
• Nodular hyperplasia = benign hepatocyte
proliferative lesion which is reversible once the
initiating agent is removed in some (but not all)
cases.

01/05/07 Dr R B Cope 128

Hepatic Neoplasia.
• Bile duct neoplasia.
• 3 types: cholangiocarcinoma (malignant),
cholangiofibroma (benign), cholangioma (benign).

• The 3 different types represent a single continuous
spectrum of lesions.

• Chemicals that induce bile duct hyperplasia usually
fail to cause bile duct neoplasia i.e. bile duct
hyperplasia is NOT a preneoplastic condition.

01/05/07 Dr R B Cope 129

Cholestasis

01/05/07 Dr R B Cope 130

Classification of Cholestasis.
• Definable at 3 levels: biochemical, physiological and
morphological.

• Biochemical cholestasis.
– Hallmark is ↑ level of bile constituents in serum i.e. ↑
conjugated bilirubin, ↑ serum bile acids.

• Physiological cholestasis.
– ↓ bile flow due to decrease in canalicular
contraction.

01/05/07 Dr R B Cope 131


• Morphological cholestasis.

– Hallmark is the accumulation of bile pigment in
canaliculi or hepatocytes, often accompanied by
deformation and/or loss of canalicular microvilli.

– Typically has a centrilobular distribution.

01/05/07 Dr R B Cope 132

Morphological cholestasis in mice chronically treated with phenobarbital.
Note the predominantly intracellular accumulation of bile pigments.
What basic mechanism does this pathology suggest? What other
changes are present? What is the distribution of this lesion?
01/05/07 Dr R B Cope 133

Gross morphology of human liver showing evidence of
cholestasis: note the color.
01/05/07 Dr R B Cope 134

• An alternative system of classification is based on the
presence or absence of evidence of damage to bile
ducts:
• Canalicular cholestasis: not associated with
destruction of cholangiocytes and therefore,
serum alkaline phosphastase (ALP) levels are
normal.
• Cholangiodestructive cholestasis/Acute bile duct
necrosis.
– Associated with ↑ serum ALP.
– Associated with destruction of cholangiocytes,
portal inflammation, bile duct proliferation and
portal fibrosis.
– Usually associated with rapid replacement of
the bile duct epithelium.
01/05/07 Dr R B Cope 135

Mechanisms of Cholestasis.
• There are at least 6 potential mechanisms of
cholestasis:

– Impaired uptake of bile precursors through the
hepatocyte basolateral cell membrane. e.g.
estrogens ↓ the Na+/K+ ATPase necessary for bile
salt transport across the hepatocyte basolateral cell
membrane.

– ↓ transcytosis of bile precursors through the
hepatocyte cytoplasm. e.g. microcystin disrupts the
hepatocyte cytoskeleton which ↓ transcytoplasmic
vesicular transport and hepatocyte deformation.

01/05/07 Dr R B Cope 136

Mechanisms of Cholestasis.
– Impaired hepatocyte apical secretion. e.g.
estrogens inhibit transport of glutathione conjugates
and bile salts.

– ↓ Canaliculus contractility.

– ↓ Integrity of bile canalicular tight junctions.

– Concentration of reactive species in the bile
canaliculus and resultant damage to cholangiocytes
and/or hepatocytes. This mechanism is probably the
most common.

01/05/07 Dr R B Cope 137

Section 3:

Rodent Liver Tumours and Human Health
Risk Assessment

01/05/07 Dr R B Cope 138


1. Understand and recognize the types of pre-neoplastic
lesions present in the rodent liver and their implications
in terms of carcinogenesis and risk assessment.
2. Understand the fundamental differences between
adenomas and carcinomas.
3. Understand the mode of action of human hepatic
carcinoma.
4. Under the ILSI/HESI mode of action framework for
interpretation of rodent liver tumour data for human risk
assessment.

01/05/07 Dr R B Cope 139

Progression to Neoplasia: Dichloroacetic Acid (DCA)

(A)Low-power photomicrograph of
an focus of hepatocellular
alteration (FHA) in a control
mouse, which is recognizable as
dysplastic under higher power
(magnification, 63; bar = 100
µm).

(B)Higher magnification of FHA in
(A) illustrating dysplasia
including nuclear enlargement,
increased nuclear/cytoplasmic
ratio, nuclear hyperchromasia,
variation in nuclear size and
shape, irregular nuclear borders,
and nucleoli that are increased in
size and number with irregular
borders (magnification, 250; bar
= 100 µm).
01/05/07 Dr R B Cope 140


(C) Large FHA in a liver from
a mouse treated with 1 g/L
DCA; note irregular border
and lack of compression at
edge (magnification, 63;
bar = 100 µm).

(D) Higher magnification of
FHA in (C) illustrating a
focus of dysplastic cells
within the LFCA
(magnification, 400; bar =
100 µm).

01/05/07 Dr R B Cope 141


(E) Edge of a large area of
dysplasia (AD) from a
mouse treated with 3.5 g/L
DCA, demonstrating
compression of adjacent
parenchyma and "pushing"
border of lesion
100 µm).

(F) Higher magnification of
AD in (E) illustrating
dysplastic cells
100 µm).

01/05/07 Dr R B Cope 142


Carcinoma

01/05/07 Dr R B Cope 143

Foci of Hepatocellular Alteration:
“Pre-neoplastic” change
• Society of Toxicologic Pathology Classifications:

• Foci of hepatocellular alteration:
• Basophilic cell foci, tigroid type and homogenous type –
increased RER and decreased cell glycogen;

• Eosinophilic (acidophilic) cell foci – deficient in glucose-6-
phosphatase; ground glass appearance;

• Clear cell foci – large unstained cytoplasm with no
vacuoles;

• Amphiphilic cell foci – intensely eosinophilic cytoplasm;

• Mixed cell foci.
01/05/07 Dr R B Cope 146

Basophilic FHA

01/05/07 Dr R B Cope 147

Eosinophilic FHA

01/05/07 Dr R B Cope 148

Clear Cell FHA

01/05/07 Dr R B Cope 149

Mixed FHA

01/05/07 Dr R B Cope 150

• Occur spontaneously with age in rats; also
occasionally in dogs & non-human primates;

• Type and number of spontaneous foci vary with strain;

• Have the characteristics of initiated ± promoted cells;

• Number increase with exposure to genotoxic
carcinogens;

• Represent an “adaptation” of the hepatocytes to a
hostile environment i.e. maladaptive response;

01/05/07 Dr R B Cope 151

• Often express placental glutathione S-transferase
(GST-P) and are UDP-glucuronosyltransferase
negative in rats. Variable expression patterns
found in mouse foci;

• Elevated replicative DNA synthesis;

• Altered expression of various growth factors;

• Over responsive to mitogens;

01/05/07 Dr R B Cope 152


• Over responsive to mitogens

• Inherent defects in growth control (i.e. becoming
autonomous in terms of growth)

• Genomic instability

• Aberrant methylation of p16 TSG

01/05/07 Dr R B Cope 153


• Mutations of ß-catenin

• Decreased apoptosis;

• Clonal origin demonstrable in vitro

01/05/07 Dr R B Cope 154

GST-P Positive FHA

01/05/07 Dr R B Cope 155

Foci of Hepatocellular Alteration: “Pre-
neoplastic” change

• Relevance to humans:

• Similar pre-neoplastic foci occur in humans exposed to
hepatic carcinogens (both viral and chemical);

• Also occur with non-genotoxic hepatocarcinogens i.e.
anabolic steroids;

• Potentially relevant to humans depending on the
mechanism/mode of action!

01/05/07 Dr R B Cope 156

• Reversibility:

• In the case of chemically stimulated FHA‟s, a high
proportion will partially or near-completely regress
when the stimulus is removed;

• Meet the criteria for “initiation + promotion”;

• Initiation is irreversible, but initiation is not
phenotypically detectable;

01/05/07 Dr R B Cope 157

FHA Versus Focal Nodular Regenerative Hyperplasia
and Nodular Regenerative Hyperplasia

• Key differences:

• Cells phenotypically normal;

• Circumscribed i.e. not invading surrounding normal
tissue;

01/05/07 Dr R B Cope 158

FHA Versus Focal Nodular Regenerative Hyperplasia
and Nodular Regenerative Hyperplasia

• Key differences:

• May be divided into pseudolobules by fibrous tissue
(focal nodular regenerative hyperplasia);

• Not pre-neoplastic.

– BUT: Can be very difficult to distinguish from
FHA!

01/05/07 Dr R B Cope 159

Foci of Pancreatic Tissue

• Metaplasia NOT neoplasia;

• Islands of seemingly “normal” exocrine pancreatic
tissue within the liver;

• Induced by Arochlor1254 i.e. Ah-receptor mediated
phemnomenon;

01/05/07 Dr R B Cope 160

Focal hepatocyte adenoma

01/05/07 Dr R B Cope 161

Adenoma Acinar Type
(An adenoma is a benign tumor (-oma) of glandular origin)

01/05/07 Dr R B Cope 162

Adenoma Trabecular Type
(An adenoma is a benign tumor (-oma) of glandular origin)

01/05/07 Dr R B Cope 164

Adenoma – Human Vs Rodent
• Rodent
• Clearly distinguishable from regenerative
hyperplasia;

• Usually larger than one lobule;

• Compress the surrounding tissue;

• Loss of normal lobular architecture but portal triads
may be present;

• Usually multifocal;

• Not encapsulated with fibrous tissue;
01/05/07 Dr R B Cope 165

Adenoma – Human Vs Rodent

• Humans
• Difficult to differentiate from regenerative
hyperplasia

• Usually solitary

• Usually encapsulated

01/05/07 Dr R B Cope 166

Carcinoma

Carcinoma: Carcinoma refers to an invasive malignant tumor
consisting of transformed epithelial cells. Alternatively, it refers to
a malignant tumor composed of transformed cells of unknown
histogenesis, but which possess specific molecular or histological
characteristics that are associated with epithelial cells, such as the
production of cytokeratins or intercellular bridges.

01/05/07 Dr R B Cope 167

Carcinoma Trabecular Type (Malignant)

01/05/07 Dr R B Cope 168

Carcinoma Acinar Type (Malignant)

What is this??

01/05/07 Dr R B Cope 169

Carcinoma Clear Cell Type (Malignant)

01/05/07 Dr R B Cope 170

Carcinoma Scirrhous Type (Malignant)

01/05/07 Dr R B Cope 171

Carcinoma Poorly Differentiated (Malignant)

01/05/07 Dr R B Cope 172

What is so important about this?

01/05/07 Dr R B Cope 173

Carcinoma – Human Vs Rodent

• Humans
• Mixed cell tumors are relatively common;
• Concurrent cirrhosis is common;
• Usually associated with chronic hepatitis;
• Rarely spontaneous – usually a history of viral
exposure and/or aflatoxin exposure and/or alcohol
exposure.

01/05/07 Dr R B Cope 174

• Rodent
• Classically metastasize to lung (why?)

• Derive from oval cells (pluripotent stem cells) in the
periportal area

• Mixed cell tumors (i.e. hepatocyte plus bile duct cell
carcinomas) do not occur

01/05/07 Dr R B Cope 175

• Rodent

• Usually do not involve concurrent cirrhosis or chronic
hepatitis

• “Spontaneous” in older animals (also in hamsters and
beagle dogs)

• “Spontaneous” tumors are common, particularly in
some strains.

01/05/07 Dr R B Cope 176

So what sort of tumor is this?

01/05/07 Dr R B Cope 177

ILSI/HESI MOA Framework

• Is the weight of evidence sufficient to establish the MOA
in animals?

• Genotoxic (classically mutagenic)?
• Potentially relevant to humans, particularly if
tumors at multiple sites;

• Nongenotoxic (non-mutagenic)?
• Relevance to humans is highly dependent on the
mechanism!

01/05/07 Dr R B Cope 178

• Are the key events in the animal MOA plausible in
humans?

• Genotoxic
• Do the mutations occur in human cells in vitro and
in vivo?
• Do the same spectrum of mutations occur?
• Is the genotoxic progression similar?

• Histopathology
• Is the same histopathological life history present in
rodents and humans?

01/05/07 Dr R B Cope 179

• Are the key events in the animal MOA plausible in humans?

• Nongenotoxic?
• Relevance is HIGHLY dependent on the mechanism;

• Do the hyperplastic effect + antiapoptotic effect occur
in humans?

• If a receptor-mediated pathway is involved, is this
pathway present in humans and of similar
pathophysiological relevance?

• Is there a clear dose threshold and what is its
relationship to human exposure?

01/05/07 Dr R B Cope 180

• Taking into account kinetic and dynamic factors, are the key
events in the animal MOA plausible in humans?

• TK is sufficiently similar to result in relevant concentrations
at the site of action?

• Promutagens activated to the same extent in humans (i.e.
TD issues)? (TD encompasses all mechanisms through
which the concentration/amount at the site of action elicits
the toxic effect);

• If redox damage is critical, does similar metabolism/events
occur in humans?

• Do the tumors occur in a non-rodent species?
01/05/07 Dr R B Cope 181

• Observation of tumours under different circumstances lends
support to the significance of the findings for animal
carcinogenicity. Significance is generally increased by the
observation of more of the following factors:

•Uncommon tumour types
•Tumours at multiple sites
•Tumours by more than one route of administration
•Tumours in multiple species, strains, or both sexes
•Progression of lesions from preneoplastic to benign to
malignant
•Reduced latency of neoplastic lesions
•Metastases (malignancy, severity of histopath)
•Unusual magnitude of tumour response
•Proportion of malignant tumours
•Dose-related increases
•Tumor promulgation following the cessation of exposure

01/05/07 Dr R B Cope 182

Relevance Depends on MOA

01/05/07 Dr R B Cope 184

Section 4:

Detection and Measurement of Liver Injury

01/05/07 Dr R B Cope 185


1. Describe and understand the methods for detection/
measurement/assessment of hepatic toxicity and
understand their advantages and limitations.

01/05/07 Dr R B Cope 186

Interpretation of Changes in Liver Absolute and
Relative Weight.
• Liver weight is strongly correlated with body weight.

• When interpreting changes, it is important to use
relative liver weight (i.e. liver to body weight ratios)
rather than absolute liver weight

• If you are using absolute liver weights, you must take
into account any changes in body weight!

01/05/07 Dr R B Cope 187

Relative Weight.
• Guidance in relation to biological significance of
changes in liver weights:

• UK PSD Guidance Document: Interpretation of Liver
Enlargement in Regulatory Toxicology Studies 2006
(http://www.pesticides.gov.uk/Resources/CRD/Migrate
d-
Resources/Documents/A/ACP_Paper_on_the_interpre
tation_of_Liver_Enlargement.pdf)

01/05/07 Dr R B Cope 188

Relative Weight.
• “The toxicological significance of a statistically
significant increase in liver weight of ≥ 10% will be
interpreted following consideration of the mechanism
of action. Findings will be interpreted as potentially
adverse, with the specific exceptions of peroxisome
proliferators and „phenobarbitone-type‟ P450 inducers”

01/05/07 Dr R B Cope 189

General Aspects of Evaluation of Liver Function.

• Tests of liver function can be used for the following:

– Detect the presence of liver disease.

– Distinguish among different types of liver disorders.

– Gauge the extent of known liver damage

– Follow the response to treatment

01/05/07 Dr R B Cope 190


• Limitations common to all tests of liver function:

– Normal results can occur in individuals with
serious liver disease (particularly near end-stage
disease).

– Liver function tests rarely provide a specific diagnosis;
rather they suggest a category of liver disease e.g.
hepatocellular or cholestatic.

01/05/07 Dr R B Cope 191


• Limitations common to all tests of liver function:

– Functional tests only measure a limited number of
hepatic functions (usually only those that are amenable
to analysis from blood samples) where as the liver
carries out thousands of biochemical functions.

– Many of the common tests do not measure liver
function; they most commonly detect cell damage
or disruption of bile flow.

– Many of the common tests are influenced by
disease outside of the liver i.e. are not absolutely
liver specific.

01/05/07 Dr R B Cope 192

Classification of Tests of Liver Function.

• Tests based on detoxification and excretory functions:

– Serum bilirubin.

– Urine bilirubin.

– Blood ammonia.

– Serum enzyme levels.

• Tests that detect cellular damage:

01/05/07 Dr R B Cope 193

Classification of Tests of Liver Function.
• Tests that measure the biosynthetic function of the liver:

– Serum albumin.

– Coagulation factors.

– Blood ammonia.


• Tests that examine liver function ex vivo.

– Liver slice cultures (experimental only).
– 3D tissue cultures
– Primary hepatocyte cultures
01/05/07 Dr R B Cope 194

Serum Bilirubin Measurement.

• Unconjugated (“indirect”) bilirubin.

– Elevation is rarely due to xenobiotic-induced primary
hepatic disease although examples of this effect do
exist.

– Mostly associated with diseases/xenobiotics that
produce hemolysis. The exceptions are heritable
defects of UDP-glucuronyltransferase and impaired
bilirubin conjugation (e.g. Gilbert‟s syndrome, Crigler-
Najjar syndrome).

01/05/07 Dr R B Cope 195


– Xenobiotics can produce an increase in serum
unconjugated bilirubin without associated hepatic injury
if they inhibit bilirubin uptake across the hepatocyte
basolateral membrane (flavispidic acid, novobiocin) or
inhibit UDP-glucuronyl transferase 1A1 (pregnanediol,
chloramphenicol and gemtamicin).

– Remember: in normal adults, the rate limiting step for
bilirubin excretion is NOT conjugation by UGT1A1. The
rate limiting step is excretion into the bile canaliculi by
MRP2! Disruption of the excretion of conjugated
bilirubin or leaking back of conjugated bilirubin from
damaged bile canaliculi/bile ducts is a far more
common xenobiotic injury than disruption of
conjugation.
01/05/07 Dr R B Cope 196


– As previously discussed the previous point is not true
for neonates who have deficient UGA1A1 and are
particularly prone to any agent that increases bilirubin
production (e.g. hemolytic agents).

01/05/07 Dr R B Cope 197


• Conjugated (“direct”) bilirubin.

– Elevated serum conjugated bilirubin almost always
implies liver or biliary tract disease.

– Elevation of serum conjugated bilirubin almost always
occurs with just about any type of liver disease.

– Prolonged elevations of serum conjugated bilirubin
result in covalent rather than reversible binding to
albumin which thus delays bilirubin clearance i.e. the
decline in serum conjugated bilirubin may be slower
than expected following severe or prolonged liver
injury.

01/05/07 Dr R B Cope 198

• Conjugated (“direct”) bilirubin.
– There are at least 2 basic causes of this phenomenon:

• “Leaking back” of conjugated bilirubin from the bile
canaliculi or bile ducts due to cholestasis, damage to
hepatocytes or bile duct epithelium (loss of tight
junctions). This is undoubtedly the most common
mechanism.

• Blockage of transport of conjugated bilirubin across
the apical hepatocyte membrane (i.e. inhibition of
MRP2). THE classical cause of this is glutathione-
conjugated sulfobromophthalein which competes for
biliary export via MRP2 but this effect occurs with
other xenobiotics. Neonates and people with Dubin-
Johnson syndrome are particularly prone to these
effects since they have relatively low levels of MRP2
01/05/07 on their apical hepatocyte cell membranes. 199

Urine Bilirubin Measurement.
• Unconjugated bilirubin is always found bound to albumin
in serum and thus does not pass through the normal renal
glomerulus. Any bilirubin found in urine is almost always
conjugated (direct) bilirubin.

• Can be measured very simply using a urine dipstick.

• Theoretically, the urine dipstick test can provide the same
information as serum bilirubin measurement, is less
invasive and almost 100% accurate.

01/05/07 Dr R B Cope 200

Blood Ammonia Measurement.

• Ammonia produced is produced in the body by protein
metabolism and by bacteria in the colon. It is detoxified by
two routes:

– In the liver by conversion to urea and subsequently
excreted by the kidneys.
– In striated muscle where it is conjugated to glutamic
acid to produce glutamine.

• Notably, patients with advanced liver disease typically
have significant muscle wasting which, in addition to the
liver failure, decreases the ability to detoxify ammonia.

01/05/07 Dr R B Cope 201

Blood Ammonia Measurement.

• Elevated blood ammonia occurs with:
– Advanced liver disease.
– Porto-systemic shunting.

• Sometimes used as an indicator of hepatic
encephalopathy.

• Problems:
– Blood ammonia levels are not correlated with the
presence or severity of hepatic encephalopathy.

– Blood ammonia levels are poorly correlated with
hepatic function.

01/05/07 Dr R B Cope 202

Blood Enzymes that Reflect Hepatocellular Damage.

• Serum enzyme assays assume that increased serum
levels are due to cellular damage, i.e. increased release
into the serum, rather than inhibition of enzyme
catabolism. Current data suggests that this is a
reasonable assumption.

• Serum enzyme levels are insensitive indicators of
hepatocellular damage.

• The absolute level of serum enzymes is not a
prognostic indicator in hepatocellular injury.

01/05/07 Dr R B Cope 203

Blood Enzymes that Reflect Hepatocellular Damage:
Alanine Aminotransferase (ALT [SGPT]).

• Primarily found in hepatocytes.

• Normally present in the serum in low concentrations and
released in high amounts with hepatocellular damage.

• Looking for a 2-3 times increase for biological
significance.

• Level is an indicator of hepatocellular membrane
damage rather than hepatocellular necrosis. Serum level
of ALT is poorly correlated with the degree of liver cell
damage.

• Usually not increased in purely cholestatic disease.
01/05/07 Dr R B Cope 204

Blood Enzymes that Reflect Hepatocellular Damage:
Aspartate Aminotransferase (AST [SGOT]).

• Primarily found in hepatocytes, cardiac muscle, skeletal
muscle, kidneys, brain, pancreas, lung, leukocytes and
erythrocytes i.e. increased AST in the absence of an
increased ALT suggests another source other than liver.

• Looking for a 2-3 times increase for biological significance

• Other features are similar to ALT.

• Level of AST in some species, e.g. horse, is of no
meaningful value.

01/05/07 Dr R B Cope 205

Blood Enzymes that Reflect Cholestasis:
Alkaline Phosphatase (ALP).

• Primarily found in or near the apical hepatocyte membranes
(i.e. the canalicular membranes).

• An increase of ALP > 4 times normal is almost always due
to cholestasis.

01/05/07 Dr R B Cope 206

Alkaline Phosphatase (ALP).

• Serum ALP consists of several isoenzymes, each of which
is tissue specific (liver, bone, placenta, small intestine).
Liver-specific isoenzyme measurement is sometimes
required, particularly if significant bone disease is present.

– Heat stability of the different isoenzmes varies: bone and
liver ALP are heat sensitive where as placental ALP is
heat stable.

– Increases in heat stable ALP strongly suggest placental
injury or the presence of an ALP producing tumor.

01/05/07 Dr R B Cope 207

Gamma Glutamyl Transpeptidase (GGT).

• Located in hepatocyte endoplasmic reticulum and in bile
duct epithelial cells.

• Blood levels of this enzyme are considered specific for
hepatic disease.

• Because of its diffuse localization in the liver, GGT is
considered less specific for cholestasis than ALP.

• Elevated levels of GGT are often interpreted to be evidence
of damage to bile duct epithelium.

01/05/07 Dr R B Cope 208

5’-nucleotidase.

• Located in or near the apical (i.e. canalicular) hepatocyte
cell membrane.

• Rarely elevated in any condition other than cholestasis and
therefore considered to be relatively specific.

01/05/07 Dr R B Cope 209

Tests Relying on Hepatic Biosynthetic Function:
Serum Albumin.
• T1/2 in serum of 15 – 20 days; 1st order kinetics with ~4%
degraded per day.

• Because of its long T1/2 and slow turnover, albumin is not
a good indicator of acute or mild hepatic dysfunction.

01/05/07 Dr R B Cope 210

Serum Albumin.
• Useful as an indicator of chronic liver disease, particularly
cirrhosis where decreases in serum albumin usually reflect
decreased albumin synthesis provided other causes of
hypoalbuminemia have been ruled out!

• Causes of hypoalbuminemia: malnutrition, protein-
loosing enteropathies and nephropathies and chronic
infections associated with sustained increases in serum
IL-1/TNF (IL-1 and TNF suppress albumin synthesis).

• Albumin measurement is only of clinical value in ~ 0.4%
of patients with liver disease!

01/05/07 Dr R B Cope 211

Coagulation Factors.
• With the exception of factor VIII, all functional clotting
factors are synthesized by the liver.

• Serum T1/2 for clotting factors ranges from 6 hours (factor
VII) to 5 days for fibrinogen.

• The most rapidly depleted clotting factor is factor VII which
is critical for the conversion of prothrombin to thrombin
during the clotting cascade (thrombin, in turn, converts
fibrinogen to fibrin monomer, the basic building block of
polymeric fibrin).

• Evidence of coagulopathy that is attributable to liver disease
is regarded as a poor prognostic sign.
01/05/07 Dr R B Cope 212

Coagulation Factors.

• The earliest detectable defect is a decline in prothrombin
time, followed sometime later by a decline in the activated
prothrombin time.

• The decline in PT is associated with the development of
clinical evidence of hemorrhage e.g. bruising, ptechial
hemorrhages etc.

• Remember, production of active factors II, VII, IX and X
require vitamin K i.e. an important differential diagnoses will
be vitamin K deficiency, warfarin treatment and
anticoagulant rodenticide poisoning.

01/05/07 Dr R B Cope 213

Tests Relying on Hepatic Metabolic Clearance:
Antipyrine, Caffeine and Galactose Clearance

• More complex to perform and more expensive than
conventional biochemical tests, but superior in monitoring
the degree of liver dysfunction.

• Involve IV injection of a compound that is mostly or
exclusively metabolized by the liver and measuring its
clearance from the circulation.

• The antipyrine clearance test is the most common and
correlates well with the degree of liver damage.

01/05/07 Dr R B Cope 214

Tests Relying on Hepatic Metabolic Clearance:
Antipyrine, Caffeine and Galactose Clearance

• The caffeine clearance test is beneficial in severe liver
lesions, but practically useless in the case of moderate
liver damage.

• The galactose clearance test can be used early in the
clinical course of jaundice to distinguish between
hepatocellular disease and biliary obstruction.

01/05/07 Dr R B Cope 215

Tests That Measure Hepatic Excretion:
Sulfobromophthalein (BSP).
• BSP is actively transported across the basolateral
hepatocyte membrane by OATP and bilitranslocase.

• BSP is conjugated to glutathione and then transported
across the apical hepatocyte membrane by MRP2.

• Competes with conjugated bilirubin for excretion by MRP2.
For both bilirubin and sulfobromophthalein, this is the rate
limiting step i.e. Like bilirubin, retention of BSP is mostly
likely due to competition or inhibition of MRP2 and impaired
transport across the hepatocyte apical cell membrane.

01/05/07 Dr R B Cope 216


• BSP tests have been largely abandoned in clinical medicine
mostly because of cost and complexity.

• They are still extensively used experimentally and are
superior to the standard biochemical tests for monitoring the
degree of liver dysfunction when significant liver damage is
present.

01/05/07 Dr R B Cope 217

• Impaired hepatic sulfobromophthalein excretion (i.e.
increased or delayed retention) has at least four potential
causes:
– Cholestasis due to impaired apical excretion (i.e.
inhibition of MRP2). This is the most likely cause since
MRP2 function is the rate-limiting step in bilirubin
excretion.

– Inhibition of glutathione-S-transferases (requires
conjugation to glutathione for excretion).

– Impaired basloateral OATP function.

– Impaired basolateral bilitranslocase function.

01/05/07 Dr R B Cope 218

Indocyanine Green (ICG).

•ICG is a water-soluble inert compound that is injected
intravenously.
•It mainly binds to albumin in the plasma. ICG is then
selectively taken up by hepatocytes via the basolateral OAT2
transporter, and subsequently excreted unchanged into the
bile via an ATP-dependent transport system.
•ICG is not metabolized; it does not undergo enterohepatic
recirculation.

01/05/07 Dr R B Cope 219

Indocyanine Green (ICG).

•ICG excretion rate in bile reflects the hepatic excretory
function and hepatic energy status.
•ICG has been found to be useful to assess liver function in
liver donors and transplant recipients, in patients with chronic
liver failure and as a prognostic factor in critically ill patients.

01/05/07 Dr R B Cope 220

Oral Cholecystographic Contrast Agents.
• Technique: a radiocontrast agent that is exclusively
excreted vial the biliary system is administered by mouth
which allows two forms of observation by CT or MRI:
• Detailed imaging of the biliary tree, particularly the
gall bladder.
• Measurement of the time required for the material to
appear in the biliary tree and to be completely cleared
from the biliary tree (measurements of biliary
excretory capacity).
•Common agents used are: iopanoic acid, sodium ipodate,
and sodium tyropanoate.
01/05/07 Dr R B Cope 221

Other Tests.
• Diagnostic imaging
• Ultrasonography, CT and MRI: high sensitivity for
detection of biliary duct changes, hepatic masses.
• Doppler CT, doppler ultrasonography and MRI
hepatic angiography can be used to assess
vasculature and hepatic hemodynamics.
• Endoscopic retrograde cholangiopancreatography
(retrograde infiltration of the biliary tract with
radiocontrast materials).

•Liver biopsy: remains the gold standard in
evaluation of liver disease in living patients.
01/05/07 Dr R B Cope 222

Section 5.

The Two Basic Classes of Hepatic Toxicants,
and Classical “Must Know” Agents Causing
Hepatic Damage.

01/05/07 Dr R B Cope 223


• Describe and understand the two basic classes of
hepatotoxicants and be able to provide examples.

• Describe and intimately understand the mechanisms of
classical Class I and Class II
hepatotoxins/hepatotoxicants.

01/05/07 Dr R B Cope 224

Class I Hepatotoxicants.
• Produce a predictable histologic pattern of hepatic
damage in most individuals within a population.

• Severity of damage is dose related.

• Damage can be reliably reproduced experimentally.

• Damage is typically fatty change, necrosis or
cholestasis.

• Damage occurs following a brief, but predictable, latent
period.

01/05/07 Dr R B Cope 225

Classical Examples of Class I Hepatotoxicants.
• Acetominophen
• Aflatoxin
• Allyl alcohol
• Bromobenzene
• Carbon tetrachloride (CCl4)
• Chloroform (HCCl3)
• Dimethylnitrosamine
• Ethanol All produce fatty
• Ethionine change or necrosis
• Orotic acid
• Phosphorus
• Tannic acid
• Tetracycline
• Thioacetamide
• Valproic acid

01/05/07 Dr R B Cope 226

Classical Examples of Class I Hepatotoxicants.
• Carbutamide
• Chlorpropamide
• Chlorpromazine
• Cyclosporine
• Erythromycin All produce
• Lantadene A (from Lantana camara) cholestasis
• Lithocholic acid. & bile duct
damage
• α-naphthylisothiocyanate.
• Manganese-billirubin.
• Methylene dianiline (Epping jaundice)
• Methyltestosterone.
• Norethandrolone.
• Sporodesmin.
01/05/07 Dr R B Cope 227

Class II Hepatotoxicants.

• Non-predictable effects on the liver.

• Idiosyncratic or hypersensitivity/immune-mediated
reactions i.e. only affect a small % of the population.

• Severity of lesion is not related to dose.

• Onset of pathology bears no consistent time relationship
to exposure.

01/05/07 Dr R B Cope 228

Class II Hepatotoxicants.

• Often signs systemic signs of allergic reactions are
present (i.e. fever, malaise, arthralgia, eosinophilia,
rash).

• Two basic mechanisms:
– immune mediated e.g. halothane.
– difference in biotransformation (rare genotype).

• Usually not detected by toxicology testing prior to the
marketing of a drug.

01/05/07 Dr R B Cope 229

Classical Examples of Class II Hepatotoxicants.
• Cincophen (antiarrhythmic agent)
• Chlorpromazine.
• Erthyromycin.
• Diclofenac.
• Halothane.
• Hydrazine MAO inhibitors (iproniazid, iscocarboxazid,
nialamic, isoniazid, phenylzine),
• Methyl DOPA.
• Indomethacin.
• Isoniazid.
• Phenylbutazone.
• Phenytoin.
• Tricrynafen (diuretic used in treatment of heart failure)
• Trimethoprim-sulfamethoxazole.
• Troglitazone (anti-diabeticDr R B Cope
01/05/07 drug). 230

Must Know Class I Hepatotoxicants:
Acetominophen.
• Mechanism:

CYP2E1
Acetominophen N-acetyl-p-benzoquinone imine
(nucleophile)

Conjugation to
Sulfonation GSH Centrilobular Necrosis

Mercapturic acid

Detoxified if Low Dose
Long-term alcohol abuse has been established as potentiating acetaminophen
toxicity via induction of CYP2E1 and depletion of glutathione. Alcoholic patients may
develop severe, even fatal, toxic liver injury after ingestion of standard therapeutic
doses of acetaminophen.

Acetominophen.
• How would the following factors affect acetominophen
toxicity?
– Prior exposure to isopropyl alcohol?
– Prior exposure to ethanol?
– Prior treatment with phenobarbital?
– Prior exposure to CCl4
– Concurrent diethylmaleate treatment (depletes GSH)?
– Concurrent treatment with piperonyl butoxide (inhibits
CYP2E1)?
– Concurrent treatment with SKF 525a?
– Exposure in cats?

01/05/07 Dr R B Cope 232

Must Know Class I Hepatotoxicants: Aflatoxin.

• Mycotoxin produced on stored grains by Aspergillus
flavus and A. parasiticus on cereal grains.
• Acute exposure results in centrilobular necrosis; hepatic
carcinogen in some species (including humans) with
chronic exposure.
• Humans are particularly resistant to acute hepatic injury
by aflatoxins; growth retardation in children and hepatic
carcinoma are THE major problems in humans.
• Mechanism of acute hepatic injury (Periportal/Zone 1):
CYP2E1 Aflatoxin epoxide
Aflatoxin B1
(Aflatoxin M1 )

Why is damage in Zone 1?
01/05/07 Dr R B Cope 233

Must Know Class I Hepatotoxicants: Allyl Alcohol.
• Important chemical precursor and common byproduct of
combustion of organic materials (including fuels).
• Produces periportal necrosis.
• Allyl alcohol is a metabolite of cyclophosphamide and is
responsible for some of this drug's effects.
• Mechanism:
ADH
Allyl alcohol Acrolein Lipid peroxidation
Depletion ofGSH
NAD+ NADH Protein damage
Why do you think that the hepatic damage due to allyl
alcohol occurs in zone I (perioportal area)?

Allyl alcohol is a suicide substrate: what do you think that
prior exposure would have on a second treatment with 234
allyl alcohol?

Amanita phalloides (Death Cap Mushroom).
• Toxins are amatoxins and phallotoxins (both are
cyclopeptides).
• Amatoxins have a propensity to concentrate in hepatocytes
because of active uptake by OAT1B3.
– Competitive substrates for OAT1B3, such as rifampicin,
have been theoretically suggested for treatment
• Amatoxins inhibit RNA polymerase II, therefore interfering
with DNA and RNA transcription
• Phallotoxins interrupt the actin polymerization-
depolymerization cycle and thus may contribute to the liver
disease due to suppression of bile canalicular motility.
• Effect is centrilobular necrosis.

01/05/07 Dr R B Cope 235

Must Know Class I Hepatotoxicants: Bromobenzene (Phenylbromide).
• ONE OF THE CLASSICAL EXAMPLES OF CYP2E1
TOXICATION
• Mechanism:

Centrilobular/Z3
necrosis
Toxicity inhibited by
inhibitors of CYP2E1
Toxicity enhanced by
inducers of CYP2E1
Toxicity enhanced by
depletors of GSH

01/05/07 Dr R B Cope 236

Must Know Class I Hepatotoxicants: Carbon Tetrachloride.
• Another of THE classical examples of CYP2E1 toxication
• Mechanism:

CYP2E1

Trichloromeithyl radical acts to:
Reacts with hydrogen to form chloroform which is then metabolized to
a radical
Reacts with itself to form hexachloroethane
Reacts with proteins -> SUICIDE SUBSTRATE FOR CYP2E1
Peroxidizes the polyenoic lipids of the endoplasmic reticulum and
triggers the subsequent generation of secondary free radicals derived
from the lipids in the membrane --> destroys the endoplasmic reticulum
resulting in decreased CYP activity and decreased protein synthesis
Triggers fatty liver by blocking the binding of triglycerol to apoproteins
01/05/07 blocking excretion of apolipoproteins from hepatocytes
thus Dr R B Cope 237

Must Know Class I Hepatotoxicants: Carbon Tetrachloride.
• Classical effect is centrilobular/zone 3 fatty change/necrosis
• CCl4 is the best studied example of the effects of modulation
of CYP levels and tissue damage.
• Potentiators of CCl4 hepatotoxicity:
– Prior exposure to any CYP2E1 inducer e.g. Ethanol, most
ketones (acetone), diabetes mellitus, isopropyl alcohol
(converted to acetone by ADH), phenobarbital
• Inhibitors of CCl4 hepatotoxicity:
– Piperonyl butoxide inhibition of CYP2E1 (remember:
initially inhibits CYP but then later induces it! Effect
depends on timing!)
– SKF525a inhibition of CYP2E1
– Concurrent treatment with a CYP2E1 substrate e.g.
concurrent treatment with ethanol, acetone

01/05/07 Dr R B Cope 238

Must Know Class I Hepatotoxicants: Chloroform.
• Classical effect is centrilobular/zone 3 fatty change/necrosis
• Mechanism:
NU = tissue nuleophile

Phosgene

01/05/07 Dr R B Cope 239

Must Know Class I Hepatotoxicants: Chloroform.

• Classical effect is centrilobular/zone 3 fatty change/necrosis:
WHY?
• Common source of human exposure is chlorinated drinking
water.
• Modulation of toxicity by modulation of CYP2E1 resembles
that of CCl4.

01/05/07 Dr R B Cope 240

Must Know Class I Hepatotoxicants: Copper.

• Several distinct diseases that all involve damage to the liver
– Acute copper poisoning.
– Wilson's disease, the Long-Evans cinnamon rat and toxic
milk mice.
– Idiopathic childhood cirrhosis and copper storage disease
in Bedlington Terriers.
– Copper toxicity in ruminants.
– Copper hepatotoxicity secondary to cholestatic defects:
Tyrolean childhood cirrhosis, Indian childhood cirrhosis,
North Ronaldsay sheep, Doberman Pinscher hepatitis, Sky
Terrier hepatitis, & non-suppurative feline
cholangioheptatitis complex.
• All require a little bit of knowledge about Cu metabolism,
storage and excretion (next couple of slides).

01/05/07 Dr R B Cope 241

GUT ENTEROCYTE CIRCULATION

CuS

S2- ?
Cu2+ hCTR1
S2-+ MoO4 2-
DMT1 ATB7A Cu2+ Albumin
RBCs
Histidine
Cu2+
Terththiomolybdate Cu-Albumin
Cu-RBC
Cu-Histidine
Storage?
Chelate

01/05/07 Dr R B Cope 242


CuMoO4-protein complex
Space of Disse Hepatocyte Bile

MoO42- + protein Ruminants in particular

Cu-Albumin
Lysosome

hCTR1 Cu2+ Cu-Atox1 ATP7b Cu2+

Albumin
Cu2+
Murr1 Cu2+
Cu-metallothionine
Murr-1-linked
Ceruloplasmin
endosome
Ceruloplasmin

ATP7b is defective in Wilson's Disease, LEC rat & toxic milk mice; Murr1
01/05/07 Dr R B Cope 243
is defective in Bedlington terriers and idiopathic childhood cirrhosis Exocytosis

Must Know Class I Hepatotoxicants: Acute Copper Toxicity.

• Remember: the primary target is the gut. Liver,
hematlogical and kidney disease will occur in those that
survive the initial GI syndrome.
• Produces centrilobular hepatic necrosis.

01/05/07 Dr R B Cope 244

Must Know Class I Hepatotoxicants: Wilson's Disease, LEC Rat,
Toxic Milk Mice.
• Humans with Wilson's disease, LEC rats and toxic milk
mice lack ATP7b and cannot synthesize ceruloplasmin
and thus accumulate copper in the liver, cornea and
CNS.
• Untreated Wilson's disease is associated with chronic
active centrilobular hepatitis and eventual cirrhosis due
to hepatic copper accumulation, damage to the cornea
and significant neuropsychiatric disease.
• Treatment is by provision of a low copper diet, the use of
copper chelators such as tetramine or penicillamine,
inclusion of ammonium tetrathiomolybdate in the diet
and increased dietary Zn, which competes with copper
for GI absorption.

01/05/07 Dr R B Cope 245

Must Know Class I Hepatotoxicants: Idiopathic Childhood
Cirrhosis and Copper Storage Disease in Bedlington Terriers.
• Both diseases are due to a defect in Murr1 which
prevents the exocytosis of endosomal copper into the
bile.
• The diseases are characterized by centrilobular chronic
active hepatitis, hepatic fibrosis and eventual death if
untreated
• In idiopathic childhood cirrhosis, storage of food items in
copper utensils, particularly the storage of acidic
materials like milk in copper containers.
• Unlike Wilson's disease, Kayser-Fleischer lines, renal
and neuropsychiatric disease does not occur in ICC or
the copper storage disease in Bedlington Terriers.
• Treatment is similar to Wilson's disease.

01/05/07 Dr R B Cope 246

Must Know Class I Hepatotoxicants: Copper Toxicity in
Ruminants.
• Disease is a cause of major livestock losses, particularly in
sheep.
• Disease is caused by a relative imbalance in the
amounts of copper and molybdenum in the diet and the
disease is better termed “chronic copper
excess/molybdenum deficiency.”
• Disease is exactly the same as molybdenum deficiency.
• Cattle, goats, swine, dogs, chickens and turkeys are
relatively resistant to this problem. The problem has
never been recorded in horses.

01/05/07 Dr R B Cope 247

Ruminants.
• Causes and factors affecting the disease:
– Consumption of any diet (grazed or compounded) with a
Cu:Mo ratio > 10:1.
– Many grazing areas in the Midwest, the Great Plains and
Central Canada contain sufficient levels of copper and low
enough levels of molybdenum to make the GRAS addition
of copper to stock feeds at the usual rate of 15 ppm
potentially toxic.
– Improved pastures containing large amounts of Trifolium
subterraneum (subterranean clover): contains little or no
Mo.
– Consumption of pastures contaminated with copper
containing pesticides/fungicides (particularly near
orchards).

01/05/07 Dr R B Cope 248

Ruminants.
• Causes and factors affecting the disease:
– Anything that impairs liver function (even when diets
containing safe levels of Cu are fed) may ↓ liver Cu
metabolism and excretion, ↑ liver Cu accumulation and
predispose to chronic Cu toxicity.
– Pyrrolizidine alkaloids are particularly important in Australia/New
Zealand.
• Heliotropium sp, Echium sp (particularly E. plantagineum;
Paterson’s curse), Senecio sp
 Lupine alkaloids

01/05/07 Dr R B Cope 249

Ruminants.
 Pathogenesis
 Essentially a 2 phase disease:
 Phase I: Characterized by the absence of disease and
chronic hepatic Cu accumulation (weeks to months).
 Phase II: Clinical disease phase characterized by
hemolytic crisis, renal failure and liver damage.

01/05/07 Dr R B Cope 250

Ruminants.
 Phase I: chronic hepatic Cu accumulation (weeks to
months)
 Disease is completely subclinical at this phase, although it
is detectable using specialized histology (see next few
slides)
 Characterized by progressive lysosomal accumulation of
copper in the liver

01/05/07 Dr R B Cope 251

Excessive copper accumulation in hepatocytes in ovine copper toxicity (rhodanine stain
for copper); excessive copper is also usually present in large amounts in hepatic Kupffer
cells. Copper can also be identified using the rubeanic acid stain. REMEMBER: LARGE
AMOUNTS OF COPPER WILL ONLY BE PRESENT IN THE LIVERS OF ASYMPTOMATIC
01/05/07
ANIMALS (I.E. BEFOREDr R B Cope HEMOLYTIC PHASE)!
THE ACUTE 252

Excessive hepatocyte coper in ovine copper toxicity (Victoria blue stain: stains
copper-associated protein); excessive copper is also usually present in large
amounts in hepatic Kupffer cells. REMEMBER: LARGE AMOUNTS OF COPPER
WILL ONLY BE PRESENT IN THE LIVERS OF ASYMPTOMATIC ANIMALS (I.E.
01/05/07 BEFORE THE ACUTE HEMOLYTIC PHASE)!
Dr R B Cope 253

Ruminants.
 Phase II: “Acute disease phase.”
 Cu levels reach a crisis point beyond which the liver cannot
excrete sufficient copper or store it in a safe manner ± other
triggering factors  hepatocellular death  immature
replacement hepatocytes are unable to rapidly absorb and
clear the excess Cu  sudden release of large amounts of
Cu into the circulation  oxidative erythrocyte cell
membrane damage and oxidation of hemoglobin to
methemoglobin  intravascular hemolysis and
methemoglobinemia  ↓ blood O2 carrying capacity 
centrilobular hepatic anoxia/necrosis  further Cu release.
 Triggers include: hepatic toxins, reduced food intake,
handling, strenuous exercise, sudden intake of Cu
containing foods, sudden cold weather or any other
stressor.
01/05/07 Dr R B Cope 254

Pale, swollen friable livers associated with the acute phase of
copper toxicity in sheep.

01/05/07 Dr R B Cope 255

Liver: Necrosis, centrilobular to submassive, with hemorrhage
due to copper toxicity in sheep
01/05/07 Dr R B Cope 256

Classical “Gunmetal Blue” kidneys from sheep with copper toxicity
01/05/07 Dr R B Cope 257

Must Know Class I Hepatotoxicants: Copper Toxicity
Secondary to Cholestatic Defects.
 Importantly, hepatic copper accumulation an associated hepatic

disease can occur secondary to just about any chronic cholestatic
condition, be it toxicant-induced or genetic.
 Classical examples of this phenomenon, all of which combine a

genetic cholestatic defect with environmental copper association
are
 Humans: Tyrolean childhood cirrhosis, Indian childhood

cirrhosis.
 Domestic animals: North Ronaldsay sheep, Doberman Pinscher

hepatitis, Sky Terrier hepatitis, & non-suppurative feline
cholangioheptatitis complex.

01/05/07 Dr R B Cope 258

Must Know Class I Hepatotoxicants: Cyclophosphamide.
• Cancer chemotherapeutic;
side-effect is severe liver
damage.
• Target is the liver
sinusoids.
• Toxicity is due to
metabolism to acrolein and
phosphoramide mustard:

259

Must Know Class I Hepatotoxicants: Endotoxin.
• Classical target is Kupffer cells due to selective accumulation.
• Endotoxins (e.g. LPS) trigger Kupffer cell activation and the
release of cytokines and reactive oxygen species which, in
turn, trigger inflammation and extensive parenchymal
damage.
• Endotoxins and Kupffer cells appear to play a key role in
ethnol-induced chronic liver disease:
– Ethanol exposure results in increased endotoxin release
and uptake by Kupffer cells.
– Endotoxin exposure appears to “prime” the liver for
damage by ethanol.
– Endotoxin is an inducer of ADH and enhances free radical
production associated with ethanol metabolism
– Endotoxin depletes GSH content in hepatocytes, reducing
the detoxification of free radicals
– Endotoxin stimulates the laying down of collagen by Ito
cells, thus favoring inappropriate repair over regeneration260

Must Know Class I Hepatotoxicants: Ethanol.
• Without question THE MAJOR CAUSE of toxic liver disease in
humans.
• In almost all cases, ethanol consumption makes all other
forms of liver disease (toxic or otherwise) worse.
Important in
ADH occurs in 3
addicts
isoforms in humans:
ADH1, ADH2, ADH3

Predominates in non-addicts

Important in
addicts

01/05/07 Dr R B Cope 261

Must Know Class I Hepatotoxicants: Ethanol Fatty Liver.

• Pathogenesis of alcohol-induced fatty liver:
– Can occur acutely after consumption of surprisingly low
amounts of ethanol over a surprisingly short period! 90-
100% of patients with alcohol hepatitis will also have
alcohol-induced fatty liver.
– There are 3 main theories regarding the pathophysiology
of ethanol-induced fatty liver:
• Decreased NAD/NADH ratio theory
• Modulation of the hypothalamic-pituitary-adrenal axis
by ethanol consumption theory.
• Inhibition of the release of VLDL into the circulation
theory.

01/05/07 Dr R B Cope 262


• Pathogenesis of alcohol-induced fatty liver:
– Decreased NAD/NADH ratio theory
• Metabolism of EtOh results in reduced amounts of
NAD+ in hepatocytes. This, in turn, is associated with
inhibition of glycerol-3-phosphate dehydrogenase (NAD
dependent), glycolysis and gluconeogenesis. Cellular
accumulation of glycerol-3-phosphate ensues which
results in enhanced esterification of fatty acids to form
triacylglycerols (neutral fats) that accumulate in
hepatocytes and a shift in metabolism towards
ketogenesis.
• Decreased NAD/NADH ratio results in decreased
availability of NAD+ for β-oxidation of fatty acids.

01/05/07 Dr R B Cope 263

• Pathogenesis of acute alcohol fatty liver:
– Modulation of the hypothalamic-pituitary-adrenal axis by
ethanol consumption theory.
• GI irritation by ethanol results in the release of arginine
vasopressin → stimulates release of ACTH due to
activation of the V1b receptor in the anterior pituitary →
↑ cortisol → increased lipid mobilization →
accumulation of fatty acids within the hepatocyte at a
rate that exceeds the capacity for β-oxidation.
– Inhibition of the release of VLDL into the circulation theory.
• Chronic ethanol consumption results in inhibition of
apolipoprotein B synthesis and decreased VLDL
synthesis and secretion by hepatocytes.

01/05/07 Dr R B Cope 264

• Centrilobular localization of steatosis results from decreased
energy stores from relative hypoxia and a shift in lipid
metabolism, along with a shift in the redox reaction caused by
the preferential oxidation of alcohol in the central zone.

01/05/07 Dr R B Cope 265


• Consequences of alcohol-induced fatty liver:
– Increased collagen turnover within the liver.
– increased propensity for cirrhosis and other fibrotic liver
diseases.
– Associated with an increased propensity for liver cancer in
humans.
– Large changes in drug metabolism by the liver, particularly
due to induction of CYP2E1
– Large changes in hormone catabolism by the liver.

01/05/07 Dr R B Cope 266

Must Know Class I Hepatotoxicants: Ethanol Hepatitis.
• Pathogenesis of alcohol hepatitis.
– There are several theories regarding the pathophysiology:
• Protein-energy malnutrition theory.
• Alterations in cell membranes theory.
• Indution of an altered metabolic state in hepatocytes
theory.
• Generation of free radicals and oxidative injury theory.
• Acetaldehyde-associated damage theory.
• The endotoxin-cytokine-Kupffer cell activation, cytokine
release and inflammation theory.
• Induction of autoimmune hepatitis theory.
• Exacerbation of hepatitis viral infections.

01/05/07 Dr R B Cope 267

• Protein-energy malnutrition theory.
– Most patients with alcoholic hepatitis exhibit evidence of
protein-energy malnutrition (PEM). In the past, nutritional
deficiencies were assumed to play a major role in the
development of liver injury. This assumption was
supported by several animal models in which susceptibility
to alcohol-induced cirrhosis could be produced by diets
deficient in choline and methionine.
– This view changed in the early 1970s after key studies by
Lieber and DiCarlo performed in baboons demonstrated
that alcohol ingestion could lead to steatohepatitis and
cirrhosis in the presence of a nutritionally complete diet.
– However, recent studies suggest that enteral or parenteral
nutritional supplementation in patients with alcoholic
hepatitis may improve survival.

01/05/07 Dr R B Cope 268

Must Know Class I Hepatotoxicants:Ethanol Hepatitis.

• Altered cell membrane theory.
– Ethanol and its metabolite, acetaldehyde, have been
shown to damage liver cell membranes.
– Ethanol can alter the fluidity of cell membranes, thereby
altering the activity of membrane-bound enzymes and
transport proteins.
– Ethanol damage to mitochondrial membranes may be
responsible for the giant mitochondria (megamitochondria)
observed in patients with alcoholic hepatitis.
– Acetaldehyde-modified proteins and lipids on the cell
surface may behave as neoantigens and trigger
immunologic injury.

01/05/07 Dr R B Cope 269

• Indution of an altered metabolic state in hepatocytes theory.
– Hepatic injury in alcoholic hepatitis is most prominent in
the centrilobular area (zone 3) of the hepatic lobule. This
zone is known to be the most dependent on anerobic
metabolism.
– The reduced NAD/NADH ratio that occurs in hepatocytes
exposed to ethanol results in inhibition of the energy-
producing steps in glycolysis. The net result is decreased
anaerobic generation of ATP in the area of the liver lobule
that is most dependent on anerobic metabolism (i.e. Zone
3).

01/05/07 Dr R B Cope 270

– Chronic alcohol consumption depresses the activity of all
mitochondrial complexes, except complex II.
– Several abnormalities in mitochondrial respiratory chain
have been described:
• Decreased activity and heme content of cytochrome
oxidase.
• Impaired electron transport and proton translocation
through complex I.
• Cecreased cytochrome b content in complex III.
• Reduced function in ATP synthase complex.
– The net result is severe impairment of mitochodrial
generation of ATP via oxidative phosphorylation.

01/05/07 Dr R B Cope 271

– Ethanol induces a hypermetabolic state in the hepatocytes,
partially because ethanol metabolism via CYP2E1 does
not result in energy capture via formation of ATP via
ethanol metabolism. Rather, this pathway leads to loss of
energy in the form of excessive heat production.
– Decreased NAD/NADH ratio also results in the inhibition of
gluconeogenesis. and inhibition of energy generation by β-
oxidation of fatty acids.

01/05/07 Dr R B Cope 272


– Due to the decreased NAD/NADH ratio, there is an
increased availability of reducing equivalents, such as
NADH, which leads to their shunting into mitochondria,
which induces the electron transport chain components to
assume a reduced state. This facilitates the transfer of an
electron to molecular oxygen to generate reactive species
as superoxide anion.
– Mitochondrial ROS generation can also derive from the
ethanol-induced changes in the mitochondrial respiratory
chain. These changes promote superoxide anion
generation within the mitochondria which leads to cell
damage and necrosis.

01/05/07 Dr R B Cope 273


– Free radicals, superoxide and hydroperoxides, are
generated as byproducts of ethanol metabolism via
CYP2E1 and catalase pathways which become
predominant in chronic alcoholism.
• CYP2E1 interacts with cytochrome reductase, which
leads to electron leaks in the respiratory chain and
ROS production. The species produced in this cascade
can interact with iron (Fenton reaction) generating even
more potent hydroxyl, ferryl and perferryl radicals which
perpetuate liver damage

01/05/07 Dr R B Cope 274


– Acetaldehyde reacts with glutathione and depletes this key
element of the hepatocytic defense against free radicals.
– Other antioxidant defenses, including selenium, zinc, and
vitamin E, are often reduced in individuals with alcoholism,
possibly due to malnutrition.

01/05/07 Dr R B Cope 275


• Acetaldehyde associated damage theory.
– Levels of acetaldehyde in the liver represent a balance
between its rate of formation (determined by the alcohol
load and activities of the 3 alcohol-dehydrogenating
enzymes) and its rate of degradation by ALDH. ALDH is
down-regulated by long-term ethanol abuse, with resultant
acetaldehyde accumulation.

01/05/07 Dr R B Cope 276


• Acetaldehyde associated damage theory.
– The deleterious effects of acetaldehyde accumulation in
hepatocytes include:
• Impaired β-oxidation of fatty acids → fatty liver and
impaired energy metabolism
• Acetaldehyde covalently binds with hepatic
macromolecules, such as amines and thiols, in cell
membranes, enzymes, and microtubules to form
acetaldehyde adducts. This binding may trigger an
immune response through formation of neoantigens,
impair function of intracellular transport through
precipitation of intermediate filaments and other
cytoskeletal elements, and stimulate Ito cells to
produce collagen.

01/05/07 Dr R B Cope 277

• The endotoxin-cytokine-Kupffer cell activation, cytokine
release and inflammation theory.

Increased endotoxin
uptake from gut

01/05/07 Dr R B Cope 278


01/05/07 Dr R B Cope 279


• Alcohol-induced autoimmune hepatitis theory.
– Active alcoholic hepatitis often persists for months after
cessation of drinking: in fact, its severity may worsen
during the first few weeks of abstinence. This observation
suggests that an immunologic mechanism may be
responsible for perpetuation of the injury.
– Levels of serum immunoglobulins, especially the
immunoglobulin A class, are increased in persons with
alcoholic hepatitis.
– Antibodies directed against acetaldehyde-modified
cytoskeletal proteins can be demonstrated in some
individuals.
– Autoantibodies, including antinuclear and anti–single-
stranded or anti–double-stranded DNA antibodies, have
also been detected in some patients with alcoholic liver
disease.
01/05/07 Dr R B Cope 280

Must Know Class I Hepatotoxicants: Methylene Dianiline.

• Famous because of “Epping Jaundice”: an outbreak of acute
cholestatic jaundice in the English city of Epping due to
consumption of bread that became contaminated with
methylene dianiline during transportation.
• MD specifically targets bile duct epithelial cells causing acute
cholestatsis.
• Mechanism is uncertain:
– Phase I N-acetylation of MD by both NAT1 and NAT2
enhances the disease and individuals that have the fast
N-acetylating NAT2 genotype are more susceptible to the
poisoning.
– Glutathione depletion enhances the disease, implying an
oxidative or free-radical-dependent mechanism

01/05/07 Dr R B Cope 281

Normal rat portal triad for comparison with the next slide. Note the healthy bile
01/05/07 duct epithelium.
Dr R B Cope 282

Portal triad of a rat treated with methylene dianiline. Note that the bile ducts
(marked with “*”) have lost their epithelium and the inflammatory infiltrate
(marked by the “►”).
01/05/07 Dr R B Cope 283

Microcystins and Nodularins.
• Cyanobacterial toxins associated with cyanobacterial blooms in water
(classically by Microcystis aeruginosa and Nodularia spumigena,, but they
are also produced by other cyanobacterial species; classically, microcystin-
LR is most associated with Microcystis cyanobacterial blooms).
• Cyclic peptides.
• Concentrate in the liver due to active uptake by OATP
• Inhibit protein phosphatases 1 and 2A which results in this leads to the
rapid disaggregation of intermediate filaments (cytokeratins) that form the
cellular scaffold. Microfilaments become detached from the cytoplasmic
membrane, which results in cell cytoskeletal deformation and bleb
formation. Cell lysis and apoptosis follow, depending on dose.
• Unusually, the necrosis is primarily midzonal (zone 2).

01/05/07 Dr R B Cope 284

Pyrrolizidine Alkaloids.
• Probably THE most important of the plant hepatotoxicants.
• On a world-wide basis, cause $billions of losses to the animal industries.
• Major epidemics of PA poisoning in humans have occurred.
• PAs are notable food contaminants, particularly in honey from hives grazed
on PA containing plants, and in grains contaminated with the seeds from PA
producing plants.
• The principal families involved are the Asteraceae (Compositae),
Boraginaceae and Leguminaceae (Fabaceae), while the main genera are
Senecio (Asteraceae), Crotalaria (Leguminaceae), Heliotroprium, Echium,
Trichodesma, and Symphytum (Boraginaceae).
• The most famous plant involved in PA poisoning of livestock in Oregon is
Tansy Ragwort (Senecio jacobaea).

01/05/07 Dr R B Cope 285

01/05/07 – Tansy Ragwort Cope
Dr R B (Senecio jacobaea). 286

• PAs are metabolized within hepatocytes to a reactive pyrrole which react
with cellular macromolecules at or near the site of formation.
– They bind most strongly with sulphydryl groups but also with amino
groups of proteins and nucleic acid bases.
• Many of the reactive pyrroles have a sufficiently long half-life to allow for
damage to the structures surrounding the hepatocytes. The sinusoidal
endothelium is particularly sensitive.
– In the case of the PAs from Crotalaria spectabilis, the reactive pyrroles
are long lived and are carried by RBCs to the lung where they induce
damage to the pulmonary vasculature

01/05/07 Dr R B Cope 287

• PA-induced hepatic disease usually takes 2 forms:
– Acute hepatic disease characterized by centrilobular necrosis and acute
hepatic failure.
– More commonly: chronic liver disease characterized by cirrhosis, veno-
occlusive disease (“peliosis hepatis”) and attempts at hepatic
regeneration.
– The development of abnormal hepatocyte megalocytes is is a
characteristic feature of the liver pathology. PAs cause alkylation of
DNA which impairs the proliferation of endogenous hepatocytes which
results in hepatocytes that enlarge (megalocytes) but cannot complete
cell division. The net result is that hepatic regeneration is ineffective.

01/05/07 Dr R B Cope 288

Must Know Class I Hepatotoxicants: Sporodesmin.

• Mycotoxin produced by Pithomyces chartarum that
grows on perennial rye grass (Lolium perenne)
• Major disease of ruminants grazed on perennial rye
grass pastures.
• Concentrates in the bile and undergoes futile redox
cycling resulting in free radical damage to the canalicular
hepatocyte cell membrane. Net result is
cholangiohepatitis and secondary photosensitization due
to phylloerythrin accumulation (facial eczema in sheep).
• Redox cycling of sporodesmin is strongly catalyzed by
copper and agents that reduce copper absorption (e.g.
Zinc) reduce the toxicity.

01/05/07 Dr R B Cope 289

Must Know Class II Hepatotoxicants: Halothane.

• Halothane is probably THE best studied human Type II
hepatotoxicant.
• There are two types of halothane toxicity:
– Type I: predominantes in rodents and is usually very
mild in humans. This is due to the formation of a
reactive metabolite.
– Type II: only occurs in humans and is very severe.
This is an autoimmune hepatitis due to neoantigen
formation.

01/05/07 Dr R B Cope 290

Must Know Class II Hepatotoxicants: Halothane.

Trifluoroacetylchloride Binds to protein

Neoantigen
formation

Autoimmune
hepatitis in humans

01/05/07 Dr R B Cope 291

Must Know Class II Hepatotoxicants: Diclofenac.

• Diclofenac is a NSAID. Similar forms of drug-induced
autoimmune hepatitis occur with many NSAIDs.
• NSAIDs act as both Type I and Type II hepatotoxicants
– Type I mechanism: due to dysregulation of
hepatocyte mitochondrial function and futile REDOX
cycling
– Type II mechanism: diclofenac metabolites form
protein adducts within the hepatocyte resulting in
neoantigen formation and immune-mediated hepatitis.

01/05/07 Dr R B Cope 292

Example of Unexpected Drug-Induced Liver Failure Detected
During Post-Market Surveillance.
FDA Public Health Advisory

Ketek (telithromycin) Tablets

(Currently being updated)

Today, January 20, 2006, Annals of Internal Medicine published an article reporting three patients who experienced serious liver toxicity following
administration of Ketek (telithromycin). These cases have also been reported to FDA MedWatch. Telithromycin is marketed and used extensively in
many other countries, including countries in Europe and Japan. While it is difficult to determine the actual frequency of adverse events from voluntary
reporting systems such as the MedWatch program, the FDA is continuing to evaluate the issue of liver problems in association with use of
telithromycin in order to determine if labeling changes or other actions are warranted. As a part of this, FDA is continuing to work to understand better
the frequency of liver-related adverse events reported for approved antibiotics, including telithromycin. While FDA is continuing its investigation of this
issue, we are providing the following recommendations to healthcare providers and patients:

Healthcare providers should monitor patients taking telithromycin for signs or symptoms of liver problems. Telithromycin should be stopped
in patients who develop signs or symptoms of liver problems.

Patients who have been prescribed telithromycin and are not experiencing side effects such as jaundice should continue taking their medicine
as prescribed unless otherwise directed by their healthcare provider.

Patients who notice any yellowing of their eyes or skin or other problems like blurry vision should contact their healthcare provider immediately.

As with all antibiotics, telithromycin should only be used for infections caused by a susceptible microorganism. Telithromycin is not effective
in treating viral infections, so a patient with a viral infection should not receive telithromycin since they would be exposed to the risk of side effects
without any benefit.
The case review in today‟s online publication by Annals of Internal Medicine reports three serious adverse events following administration of
telithromycin. All three patients developed jaundice and abnormal liver function. One patient recovered, one required a transplant, and one died. When
the livers of the latter two patients were examined in the laboratory, they showed massive tissue death. These two patients had reported some alcohol
use. All three patients had previously been healthy and were not using other prescription drugs. The FDA is also aware that these patients were all
treated by physicians in the same geographic area. The significance of this observation is not clear at the present time.

In pre-marketing clinical studies, including a large safety trial and data from other countries, the occurrence of liver problems was infrequent and
usually reversible. Based on the pre-marketing clinical data, it appeared that the risk of liver injury with telithromycin was similar to that of other
marketed antibiotics. Nonetheless, the product label advises doctors about the potential for liver-related adverse events associated with the use of
telithromycin.

Telithromycin is an antibiotic of the ketolide class. It was the first antibiotic of this class to be approved by the FDA in April, 2004 for the treatment of
respiratory infections in adults caused by several types of susceptible microorganisms including Streptococcus pneumoniae and Haemophilus
influenzae.
01/05/07 Dr R B Cope 293

Example of Unexpected Drug-Induced Liver Failure Detected

Approx 50% of telithromycin is metabolized
to an inactive metabolite by CYP3A4.

Question: is this yet another case of phase I
toxication or is another mechanism
involved?

01/05/07 Dr R B Cope 294

Examples of Unexpected Drug-Induced Liver Failure Detected

• Other recent examples (2006 and 2007 )of
unexpected drug-induced liver toxicity that was
detected by post-market surveillance:
• Ketek (telithromycin)
• Cymbalta (duloxetine hydrochloride)
• Betaseron (interferon beta-1b)
• Viramune (nevirapine)
• Serzone (nefazodone hydrochloride)
• Kava kava (“natural” health supplement i.e. regulated
as a food and not a drug).
• Arava (leflunomide)
01/05/07 Dr R B Cope 295

Unexpected Drug-Induced Liver Failure Detected During Post-
Market Surveillance.
• Why so many?
• Idiosyncratic reactions:
• Currently there is no possible way to test a large enough
number of animals or humans to ensure that every genotype
is examined.
• Use of inbred (i.e. syngenomic) strains in pre-market testing.
• Economic pressure: tendency to ignore or “weasel word” a
way around the one or two patients that have severe toxic
reactions during the clinical trials (as was the case with
telithromycin) due to the high costs of bringing a new drug to
market (total development costs from concept to market is
now approaching $US1 billion and 10 years of work for each
new molecule!)
• Toxicogenomics has reduced this problem, but it is not
perfect: not every single allele has been sequenced let alone
incorporated onto a gene chip.
01/05/07 Dr R B Cope 296

The Latest Lawsuit Due to Unexpected Drug-Induced Liver
Failure Detected During Post-Market Surveillance.
• Why so many?
• Immune-mediated reactions:
• Current techniques are good at detecting strong
sensitizers but are very poor at detecting marginal
or weak sensitizers that sometimes take months
or years of exposure before the immune reaction
is manifested.
• There is currently no way to test every Ig idiotype
or T-cell receptor type present in the entire human
population for reactivity with a new drug (although
there are some obvious guidelines e.g. avoid
molecules or metabolites that have covalent
binding to host proteins i.e. behave like hapten-
carriers).
01/05/07 297
Dr R B Cope

Section 6:

Mode of Action of Rodent Forestomach Tumours:
Relevance to Humans.


1. Under the ILSI/HESI mode of action framework for
interpretation of stomach tumour data for human risk
assessment.

01/05/07 Dr R B Cope 299

Gross anatomy of murine forestomach after NMBA
(N-nitrosomethylbenzylamine) treatment.

Zanesi N et al. PNAS 2001;98:10250-10255

©2001 by The National Academy of Sciences

NMBA-induced histopathology of murine forestomach.

Zanesi N et al. PNAS 2001;98:10250-10255

©2001 by The National Academy of Sciences

Introduction
• Forestomach tumors/pre-neoplastic lesions in rats and
mice are a common finding in repeat-dose toxicology
studies;

• Debate over the human relevance due to:
• Dose and exposure differences between rodents and
humans;
• Substantial toxicokinetic differences (exposure);
• Substantial anatomical differences;
• Substantial physiological/metabolic differences of the
forestomach epithelium;
• Different mechanisms and tumor types in humans
compared with rodents;

Dose and Exposure Problems
• Doses used in rodent oral carcinogenesis often far exceed
normal human environmental exposure conditions
(possible rare exception is some direct food additives);

• Doses that produce forestomach irritation in rodents really
should be considered as exceeding the MTD – i.e. poor
practice in rodent carcinogenesis studies and not according
to GLP/test guidelines;

Dose and Exposure Problems

• Gavage can produce forestomach irritation and is not
physiological:
– Large volumes;
– Damage to the mucosa;
– Esophageal reflux;
– Possibly replicates tablets (but not capsules);

Tissue specificity
• Forestomach carcinogens divisible into at least 3
categories:

– Produce forestomach tumors and tumors at other sites
when administered by gavage;
– Produce only forestomach tumors when administered by
gavage;
– Produce forestomach tumors and tumors when
administered by non-oral routes;

• In terms of human relevance, forestomach + tumors at
other sites is likely to be more important except in the case
of site of first contact carcinogens.

Tissue concordance/anatomical issues
• Humans do not have a forestomach or a pars esophagea:

– Roughly equivalent tissue in terms of histology is the
esophagus;
– Humans do not store food in the esophagus where as
rodents store food in the forestomach;
– Transit time through the human stomach is lower than
transit time through the rodent stomach (forestomach)
 difference in tissue exposure;
– Chemicals pass quickly through the human esophagus
and thus the exposure is very limited compared with
chemical exposure of the rodent forestomach.

Tissue concordance/anatomical issues
• Physiological issues:

– Rodent forestomach does not have a protective mucous
coating  increased tissue exposure to chemicals and
more prone to irritant effects;

– pH in rodent forestomach is higher than the pH of the
human stomach  relevant to detoxification (e.g.
hexavalent chromium to trivalent chromium in low pH of
human stomach);

– Potential metabolic differences of rodent forestomach
epithelium  conversion of 2-butoxy ethanol to 2-
butoxyacetic acid in rodent forestomach but not in
human stomach;

Tumour types and biology issues
• Rodents
– Predominant tumor types are papillomas (non-
malignant) and squamous cell (low malignancy –
regional metastasis) carcinomas;

– Typically located at the limiting ridge;

– Possibly have some relevance to human esophageal
squamous cell carcinoma BUT chemical exposure of the
human esophagus is much lower than in the rodent
forestomach due to much lower transit time (no storage
in esophagus);
– Not relevant to human esophageal adenocarcinoma.

Tumour Types and Biology Issues

• Humans

– All human stomach cancers are gastric
adenocarcinomas and arise from the glandular
epithelium;

– Rodent forestomach tumors have a different
histiogenesis and are not relevant to the human gastric
tumors;

Genotoxicity Issues
• Forestomach carcinogens are divisible into 2 basic groups:

– DNA reactive chemicals (classical in vivo genotoxic
carcinogens)
• Site of first contact carcinogens (generally direct
acting carcinogens and are usually highly reactive
chemicals; typically direct acting alkylating agents);
• Classical pro-carcinogen DNA reactive chemicals;

– Non-DNA reactive chemicals (classical non-genotoxic
carcinogens);
• Typically irritant chemicals or chemicals that produce
local increased cell turnover.

Genotoxicity Issuses
• Site of first contact carcinogens:
– Generally require no metabolism to be carcinogenic;
– Generally will produce tumors at other sites if the route
of administration is different  tumor location is the site
of contact;
– Generally only produce forestomach tumors in
gavage/dietary studies because of limited/no systemic
bioavailability;
– Typically alkylating agents;
– Typically genotoxicants in vitro and in vivo;

– Forestomach tumours are potentially human
relevant but only at the site of first contact in
humans (e.g. dermal exposures)

Genotoxicity Issuses
• Classical pro-carcinogen DNA reactive chemicals;
– Generally pro-carcinogens;
– Often produce tumours at more than one anatomical site
following oral dosing (at least one systemic site +
forestomach);
– Often other routes of administration also result in
tumors;
– Generally systemically bioavailable;

– Human relevance of forestomach tumors depends
on: (a) was there evidence of gastric irritation; (b)
were the doses excessive (> MTD); (c) were the
effects only seen with gavage dosing/diet studies
and not with drinking water studies?

• Observation of tumours under different circumstances lends support
to the significance of the findings for animal carcinogenicity.
Significance is generally increased by the observation of more of the
following factors:

•Uncommon tumour types;
•Tumours at multiple sites;
•Tumours by more than one route of administration;
•Tumours in multiple species, strains, or both sexes;
•Progression of lesions from preneoplastic to benign to
malignant;
•Reduced latency of neoplastic lesions;
•Metastases (malignancy, severity of histopath);
•Unusual magnitude of tumour response;
•Proportion of malignant tumours;
•Dose-related increases;
•Tumor promulgation following the cessation of exposure.

Benzo(a)pyrene (IARC 1)
Parameter
Genotoxicity in vivo that is relevant to humans +
Forestomach cancers following oral dosing +
Not observed in drinking water studies, only observed with gavage/diet studies -
Only observed at doses that irritate the forestomach (> MTD) -
Uncommon tumour types; +
Tumours at multiple sites; +
Tumours by more than one route of administration; +
Tumours in multiple species, strains, or both sexes; +
Progression of lesions from preneoplastic to benign to malignant; +
Reduced latency of neoplastic lesions; +
Metastases (malignancy, severity of histopath); +
Unusual magnitude of tumour response; +
Proportion of malignant tumours; +
Dose-related increases; +
Tumour promulgation following the cessation of exposure. +

Ethyl Acrylate

•Oral gavage: dose related increases in the incidence of
squamous-cell papillomas and carcinomas of the
forestomach were observed in rats and mice. Exposure
caused gastric irritancy;

•Ethyl acrylate was tested by inhalation in the same
strains of mice and rats; no treatment-related neoplastic
lesions were observed;

•No treatment-related tumour was observed following
skin application of ethyl acrylate for lifespan to male
mice.

Ethyl acrylate (IARC 2B)
Parameter
Genotoxicity in vivo that is relevant to humans -
Not observed in drinking water studies, only observed with gavage/diet studies ?
Only observed at doses that irritate the forestomach (> MTD) +
Uncommon tumour types; -
Tumours at multiple sites; -
Tumours by more than one route of administration; -
Tumours in multiple species, strains, or both sexes; +
Progression of lesions from preneoplastic to benign to malignant; +
Reduced latency of neoplastic lesions; +
Metastases (malignancy, severity of histopath); -
Unusual magnitude of tumour response; -
Proportion of malignant tumours; -
Dose-related increases; -
Tumour promulgation following the cessation of exposure. +

Mercuric chloride (IARC 3)
Parameter
Genotoxicity in vivo that is relevant to humans -
Not observed in drinking water studies, only observed with gavage/diet ?
studies
Only observed at doses that irritate the forestomach (> MTD) +
Uncommon tumour types; -
Tumours at multiple sites; -
Tumours by more than one route of administration; (thyroid follicular cell adenomas)

Tumours in multiple species, strains, or both sexes; -
Progression of lesions from preneoplastic to benign to malignant; -
Reduced latency of neoplastic lesions; -
Metastases (malignancy, severity of histopath); -
Unusual magnitude of tumour response; -
Proportion of malignant tumours; -
Dose-related increases; -
Tumour promulgation following the cessation of exposure. -

Section 7:

Case Studies

01/05/07 Dr R B Cope 323

Liver nicnas-nov-2012

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