Nursing assessment and management of patients with hepatic disorders
Liver nicnas-nov-2012
1. An Introduction to the Toxicology of the Liver
& Rodent Stomach.
Rhian B. Cope BVSc BSc(Hon 1) PhD DABT ERT
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2. 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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3. 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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4. 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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5. 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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6. 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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7. Structure of the Liver Lobule.
Low magnification view ofBthe a liver lobule in the pig
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8. Structure of the Liver Lobule.
01/05/07 Low magnification view B Cope human liver lobule
Dr R of the 8
11. Structure of the Liver Lobule.
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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17. Structure of the Liver Acinus.
• Acinar zone 1 approximates “Periportal” using the
“Lobular” system.
• Acinar zone 3 approximates “Centrilobular” using the
“Lobular” system.
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18. Describe the distribution of damage (necrosis) in this liver
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section using the “lobular” and “acinar” system.
19. ?
Describe the distributionDr R B Cope
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of damage (necrosis) in this liver
19
section using the “lobular” and “acinar” system.
20. Central Vein
Describe the distributionDr R B Cope
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of damage (necrosis) in this liver
20
section using the “lobular” and “acinar” system.
22. Structure of the Liver Acinus.
• 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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23. Structure of the Liver Acinus.
• All hepatocytes are NOT equal. Important
functional/physiological differences occur between
hepatocytes in different acinar zones.
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24. 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
25. 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
26. 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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27. 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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29. Pit Cells.
• Located in the space of Disse.
• Function as NK or LAK cells.
• Important in inflammation.
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30. 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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31. 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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34. 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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35. Bile Formation and Hepatic Excretion.
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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36. Bile Formation and Hepatic Excretion.
• 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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37. Major Hepatocyte and Cholangiocyte
Transporters involved in Bile Formation
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38. 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.
39. 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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40. 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)
41. 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
42. 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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43. 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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44. 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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45. Important Aspects of Bilirubin Excretion.
• 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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46. Important Aspects of Bilirubin Excretion.
• 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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47. 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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48. Bilirubin Excretion in the Neonate.
• 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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49. Bilirubin Excretion in the Neonate.
• 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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51. 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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52. Basis for the Susceptibility of the Liver to Toxicity.
• 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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53. Basis for the Susceptibility of the Liver to Toxicity.
• 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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54. 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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55. 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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56. 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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58. 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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59. Ascites following severe liver disease. Note the eversion of
the umbilicus.
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60. Mid-level abdominal CT scans. Left = normal; Right = ascites
secondary to liver failure.
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61. Clinical Signs of Advanced or Chronic
Hepatocellular Disease.
• 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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62. Caput medusae associated with portal hypertension,
portosystemic shunting and severe liver disease.
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63. Clinical Signs of Advanced or Chronic
Hepatocellular Disease.
• 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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64. Clinical Signs of Advanced or Chronic
Hepatocellular Disease.
• 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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65. 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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66. 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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68. Severe secondary photosensitzation of the udder of a cow
with advanced hepatic disease (again due to sporodesmin)
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69. Section 2:
Responses of the Liver to Toxic Injury.
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70. Learning Tasks Section 2.
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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71. 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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72. 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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73. Hepatocellular Adaptive Responses.
• 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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74. Hepatocellular Adaptive Responses.
• Represent adaptive responses to xenobiotic response
rather than hepatocellular damage per se.
• Used as histological markers of xenobiotic exposure.
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75. Hepatocellular Adaptive Responses.
• 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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76. 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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77. Hepatocellular Adaptive Responses:
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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79. 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
80. Hepatocellular Adaptive Responses:
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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81. Hepatocellular Adaptive Responses:
Eosinophilic Centrilobular Hepatocellular Hypertrophy.
• Reversible following removal of the initiating agent.
• 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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82. 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
83. Hepatocellular Adaptive Responses:
Xenobiotic-Induced Hepatocyte Hyperplasia.
• Usually accompanied by CYP induction,
hepatomegaly, and hepatocyte hypertrophy.
• Never continues for more than a few days.
• Reversible following removal of the initiating agent.
Reversion is associated with ↑ hepatocyte apoptosis.
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84. Derived from the UK PSD guideline
(included as an appendix to the
notes)
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85. 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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86. 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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87. 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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89. Reversible Hepatocellular Injury:
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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90. Reversible Hepatocellular Injury:
Hepatic Lipidosis (“Fatty Liver”).
• 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.
01/05/07 Dr R B Cope 90
91. Reversible Hepatocellular Injury:
Hepatic Lipidosis (“Fatty Liver”).
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.
01/05/07 Dr R B Cope 91
92. Reversible Hepatocellular Injury:
Hepatic Lipidosis (“Fatty Liver”).
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
93. Human liver. Fatty change due to alcohol. Note the color.
Surface will feel “greasy”.
01/05/07 93
94. Hepatocyte fatty change due to ethanol exposure. Note: fat
droplets appear clear due to their extraction during tissue
01/05/07
processing. 94
95. Fine needle aspirates of hepatocytes. Normal on
01/05/07 the left, fatty change on the right. 95
96. 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
97. Hepatocellular Death:
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
98. Hepatocellular Death:
Centrilobular, Zone 3 or Periacinar Necrosis.
• 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
99. Hepatocellular Death:
Centrilobular, Zone 3 or Periacinar Necrosis.
• 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
102. Hepatocellular Death:
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
103. Hepatocellular Death:
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
105. Hepatocellular Death:
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
106. Hepatocellular Death:
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
107. Hepatic massive necrosis. Note the periportal
accumulation of bile pigments.
01/05/07 Dr R B Cope 107
109. 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
110. 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
111. 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
112. 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
113. 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
114. 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
116. 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
117. 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
118. 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
119. 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
120. 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
121. 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
122. Hepatocellular Pigmentation.
• 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
123. Hepatocellular Pigmentation.
• 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
124. 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
125. 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
126. Oval cell hyperplasia in a mouse exposed to a hepatic
carcinogen.
01/05/07 Dr R B Cope 126
127. 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
128. 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
129. 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
131. 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
132. Classification of Cholestasis.
• 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
133. 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
134. Gross morphology of human liver showing evidence of
cholestasis: note the color.
01/05/07 Dr R B Cope 134
135. Classification of Cholestasis.
• 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
136. 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
137. 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
138. Section 3:
Rodent Liver Tumours and Human Health
Risk Assessment
01/05/07 Dr R B Cope 138
139. Learning Tasks Section 3.
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
140. 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
141. Progression to Neoplasia: Dichloroacetic Acid (DCA)
(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
142. Progression to Neoplasia: Dichloroacetic Acid (DCA)
(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
(magnification, 63; bar =
100 µm).
(F) Higher magnification of
AD in (E) illustrating
dysplastic cells
(magnification, 400; bar =
100 µm).
01/05/07 Dr R B Cope 142
151. Foci of Hepatocellular Alteration:
“Pre-neoplastic” change
• 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
152. Foci of Hepatocellular Alteration:
“Pre-neoplastic” change
• 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
153. Foci of Hepatocellular Alteration:
“Pre-neoplastic” change
• 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
154. Foci of Hepatocellular Alteration:
“Pre-neoplastic” change
• Mutations of ß-catenin
• Decreased apoptosis;
• Clonal origin demonstrable in vitro
01/05/07 Dr R B Cope 154
156. 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
157. Foci of Hepatocellular Alteration:
“Pre-neoplastic” change
• 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
158. 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
159. 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
160. 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
165. 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
166. Adenoma – Human Vs Rodent
• Humans
• Difficult to differentiate from regenerative
hyperplasia
• Usually solitary
• Usually encapsulated
01/05/07 Dr R B Cope 166
167. 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
173. What is so important about this?
01/05/07 Dr R B Cope 173
174. 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
175. Carcinoma – Human Vs Rodent
• 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
176. Carcinoma – Human Vs Rodent
• 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
177. So what sort of tumor is this?
01/05/07 Dr R B Cope 177
178. 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
179. ILSI/HESI MOA Framework
• 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
180. ILSI/HESI MOA Framework
• 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
181. ILSI/HESI MOA Framework
• 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
182. • 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
185. Section 4:
Detection and Measurement of Liver Injury
01/05/07 Dr R B Cope 185
186. Learning Tasks Section 4.
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
187. 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
188. Interpretation of Changes in Liver Absolute and
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
189. Interpretation of Changes in Liver Absolute and
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
190. 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
191. General Aspects of Evaluation of Liver Function.
• 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
192. General Aspects of Evaluation of Liver Function.
• 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
193. 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:
– Serum enzyme levels.
01/05/07 Dr R B Cope 193
194. Classification of Tests of Liver Function.
• Tests that measure the biosynthetic function of the liver:
– Serum albumin.
– Coagulation factors.
– Blood ammonia.
– Serum enzyme levels.
• 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
195. 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).
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196. Serum Bilirubin Measurement.
• Unconjugated (“indirect”) bilirubin.
– 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.
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197. Serum Bilirubin Measurement.
• Unconjugated (“indirect”) bilirubin.
– 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).
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198. Serum Bilirubin Measurement.
• 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.
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199. Serum Bilirubin Measurement.
• 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
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200. 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.
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201. 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.
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202. 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.
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203. 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.
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204. 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.
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205. 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.
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206. 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.
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207. Blood Enzymes that Reflect Cholestasis:
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.
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208. Blood Enzymes that Reflect Cholestasis:
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.
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209. Blood Enzymes that Reflect Cholestasis:
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.
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210. 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.
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211. Tests Relying on Hepatic Biosynthetic Function:
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!
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212. Tests Relying on Hepatic Biosynthetic Function:
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.
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213. Tests Relying on Hepatic Biosynthetic Function:
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.
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214. 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.
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215. 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.
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