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




01/05/07                    Dr R B Cope                 1
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.

 01/05/07               Dr R B Cope                     2
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.


01/05/07                           Dr R B Cope                               3
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.




01/05/07                 Dr R B Cope                    4
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.

     01/05/07                        Dr R B Cope                                 5
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?

01/05/07                   Dr R B Cope                              6
Structure of the Liver Lobule.




     Low magnification view ofBthe a liver lobule in the pig
01/05/07                   Dr R Cope                           7
Structure of the Liver Lobule.




01/05/07   Low magnification view B Cope human liver lobule
                               Dr R of the                    8
Structure of the Liver Lobule.




01/05/07               Dr R B Cope          9
Structure of the Liver Lobule.




01/05/07              Dr R B Cope           10
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?




  01/05/07                  Dr R B Cope        11
Structure of the Liver Acinus.




01/05/07              Dr R B Cope           12
Structure of the Liver Acinus.




01/05/07               Dr R B Cope          13
Structure of the Liver Acinus.




01/05/07              Dr R B Cope           14
Structure of the Liver Acinus.




01/05/07               Dr R B Cope          15
01/05/07   Dr R B Cope   16
Structure of the Liver Acinus.


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



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




01/05/07                 Dr R B Cope                     17
Describe the distribution of damage (necrosis) in this liver
01/05/07                     Dr R B Cope                      18
         section using the “lobular” and “acinar” system.
?




   Describe the distributionDr R B Cope
01/05/07
                             of damage (necrosis) in this liver
                                                              19
       section using the “lobular” and “acinar” system.
Central Vein



   Describe the distributionDr R B Cope
01/05/07
                             of damage (necrosis) in this liver
                                                              20
       section using the “lobular” and “acinar” system.
Central Vein



01/05/07
           Centrilobular orB Zone 3 Necrosis.
                       Dr R Cope                21
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.




   01/05/07                 Dr R B Cope                     22
Structure of the Liver Acinus.



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




 01/05/07                  Dr R B Cope                 23
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.




01/05/07                   Dr R B Cope                      26
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.


01/05/07                    Dr R B Cope                     27
Pigment accumulation within Kupffer cells.
01/05/07                    Dr R B Cope                 28
Pit Cells.
• Located in the space of Disse.

• Function as NK or LAK cells.

• Important in inflammation.




01/05/07                  Dr R B Cope   29
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.




01/05/07                       Dr R B Cope                     30
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?

 01/05/07                         Dr R B Cope                            31
A Concise Summary of Key Hepatic Functions




01/05/07                                  32
Consequences of Disruption of Hepatic Function
                                    Consequences




                                              33
01/05/07
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


01/05/07                        Dr R B Cope                               34
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.



01/05/07                       Dr R B Cope                             35
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.




01/05/07                    Dr R B Cope                         36
Major Hepatocyte and Cholangiocyte
           Transporters involved in Bile Formation




01/05/07                   Dr R B Cope               37
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




 01/05/07                       Dr R B Cope                           39
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




01/05/07                  Dr R B Cope                      42
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).




01/05/07                  Dr R B Cope                      43
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.
 01/05/07                         Dr R B Cope                             44
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.




01/05/07                   Dr R B Cope                           45
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.




01/05/07                  Dr R B Cope                   46
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.



01/05/07                  Dr R B Cope                       47
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.



01/05/07                  Dr R B Cope                        48
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.

01/05/07                     Dr R B Cope                 49
Globus pallidus staining with bilirubin
01/05/07                  Dr R B Cope                50
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.

 01/05/07                   Dr R B Cope                       51
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.
 01/05/07                    Dr R B Cope                         52
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)
     01/05/07                   Dr R B Cope                         53
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!

01/05/07   Dr R B Cope                                  54
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).




01/05/07                   Dr R B Cope                     55
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.


01/05/07                   Dr R B Cope                   56
01/05/07   Dr R B Cope   57
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).

01/05/07                  Dr R B Cope                     58
Ascites following severe liver disease. Note the eversion of
                       the umbilicus.
01/05/07                  Dr R B Cope                     59
Mid-level abdominal CT scans. Left = normal; Right = ascites
                 secondary to liver failure.

 01/05/07                 Dr R B Cope                    60
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).
01/05/07                            Dr R B Cope                                    61
Caput medusae associated with portal hypertension,
       portosystemic shunting and severe liver disease.
01/05/07                   Dr R B Cope                     62
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).

01/05/07                  Dr R B Cope                      63
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.




01/05/07                  Dr R B Cope                 64
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)

01/05/07                           Dr R B Cope                       65
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.”

01/05/07                   Dr R B Cope                     66
Secondary photosensitization of the face due to
01/05/07      sporodesmin poisoning in a sheep
                           Dr R B Cope                     67
Severe secondary photosensitzation of the udder of a cow
     with advanced hepatic disease (again due to sporodesmin)
01/05/07                    Dr R B Cope                         68
Section 2:

           Responses of the Liver to Toxic Injury.




01/05/07                 Dr R B Cope                 69
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.




01/05/07                    Dr R B Cope                     70
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.

   01/05/07                   Dr R B Cope                     71
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.

01/05/07                    Dr R B Cope                         72
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!




01/05/07                     Dr R B Cope                        73
Hepatocellular Adaptive Responses.


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

•     Used as histological markers of xenobiotic exposure.




01/05/07                   Dr R B Cope                       74
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.




01/05/07                    Dr R B Cope                    75
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.




01/05/07                    Dr R B Cope                     76
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).




01/05/07                    Dr R B Cope                       77
Centrilobular hepatocyte hypertrophy in a mouse treated
              with phenobarbital for 8 months.
01/05/07                 Dr R B Cope                    78
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
 01/05/07
                CYP (particularly CYP2E1) induction.             79
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.




 01/05/07                   Dr R B Cope                      80
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

 01/05/07                     Dr R B Cope                       81
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.
01/05/07                                                                           82
                                       Dr R B Cope
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.




01/05/07                   Dr R B Cope                        83
Derived from the UK PSD guideline
                         (included as an appendix to the
                         notes)




01/05/07   Dr R B Cope                              84
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.

01/05/07                   Dr R B Cope                       85
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.


01/05/07                   Dr R B Cope                     86
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.

01/05/07                Dr R B Cope            88
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



01/05/07                   Dr R B Cope                      89
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
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
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
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
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
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
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
Centrilobular necrosis.
01/05/07          Dr R B Cope        100
Hepatic centrilobular necrosis.
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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
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
Periportal degeneration and portal cirrhosis.
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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
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
Hepatic massive necrosis. Note the periportal
                  accumulation of bile pigments.
01/05/07                      Dr R B Cope                  107
Hepatic massive necrosis.
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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.




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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.

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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




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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.


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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
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.
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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
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.
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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.


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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.




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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.




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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
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
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
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
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
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
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
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
Progression to Neoplasia: Dichloroacetic Acid (DCA)




                              Carcinoma




01/05/07                Dr R B Cope                   143
01/05/07   Dr R B Cope   144
01/05/07   Dr R B Cope   145
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
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;

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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
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




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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
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!


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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
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;




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Focal hepatocyte adenoma




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




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01/05/07   Dr R B Cope   163
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
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
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
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
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
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
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
• 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
01/05/07   Dr R B Cope   183
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
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
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
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
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
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
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
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
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
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
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
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.
    01/05/07                        Dr R B Cope                      196
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).




    01/05/07                        Dr R B Cope                      197
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.

    01/05/07                       Dr R B Cope                      198
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
    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
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.




    01/05/07                       Dr R B Cope                     207
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.


    01/05/07                    Dr R B Cope                        208
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.




    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
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!


    01/05/07                       Dr R B Cope                    211
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.
    01/05/07                     Dr R B Cope                         212
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.


    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
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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 01/05/07 Dr R B Cope 1
  • 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. 01/05/07 Dr R B Cope 2
  • 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. 01/05/07 Dr R B Cope 3
  • 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. 01/05/07 Dr R B Cope 4
  • 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. 01/05/07 Dr R B Cope 5
  • 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? 01/05/07 Dr R B Cope 6
  • 7. Structure of the Liver Lobule. Low magnification view ofBthe a liver lobule in the pig 01/05/07 Dr R Cope 7
  • 8. Structure of the Liver Lobule. 01/05/07 Low magnification view B Cope human liver lobule Dr R of the 8
  • 9. Structure of the Liver Lobule. 01/05/07 Dr R B Cope 9
  • 10. Structure of the Liver Lobule. 01/05/07 Dr R B Cope 10
  • 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? 01/05/07 Dr R B Cope 11
  • 12. Structure of the Liver Acinus. 01/05/07 Dr R B Cope 12
  • 13. Structure of the Liver Acinus. 01/05/07 Dr R B Cope 13
  • 14. Structure of the Liver Acinus. 01/05/07 Dr R B Cope 14
  • 15. Structure of the Liver Acinus. 01/05/07 Dr R B Cope 15
  • 16. 01/05/07 Dr R B Cope 16
  • 17. Structure of the Liver Acinus. • Acinar zone 1 approximates “Periportal” using the “Lobular” system. • Acinar zone 3 approximates “Centrilobular” using the “Lobular” system. 01/05/07 Dr R B Cope 17
  • 18. Describe the distribution of damage (necrosis) in this liver 01/05/07 Dr R B Cope 18 section using the “lobular” and “acinar” system.
  • 19. ? Describe the distributionDr R B Cope 01/05/07 of damage (necrosis) in this liver 19 section using the “lobular” and “acinar” system.
  • 20. Central Vein Describe the distributionDr R B Cope 01/05/07 of damage (necrosis) in this liver 20 section using the “lobular” and “acinar” system.
  • 21. Central Vein 01/05/07 Centrilobular orB Zone 3 Necrosis. Dr R Cope 21
  • 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. 01/05/07 Dr R B Cope 22
  • 23. Structure of the Liver Acinus. • All hepatocytes are NOT equal. Important functional/physiological differences occur between hepatocytes in different acinar zones. 01/05/07 Dr R B Cope 23
  • 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. 01/05/07 Dr R B Cope 26
  • 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. 01/05/07 Dr R B Cope 27
  • 28. Pigment accumulation within Kupffer cells. 01/05/07 Dr R B Cope 28
  • 29. Pit Cells. • Located in the space of Disse. • Function as NK or LAK cells. • Important in inflammation. 01/05/07 Dr R B Cope 29
  • 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. 01/05/07 Dr R B Cope 30
  • 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? 01/05/07 Dr R B Cope 31
  • 32. A Concise Summary of Key Hepatic Functions 01/05/07 32
  • 33. Consequences of Disruption of Hepatic Function Consequences 33 01/05/07
  • 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 01/05/07 Dr R B Cope 34
  • 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. 01/05/07 Dr R B Cope 35
  • 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. 01/05/07 Dr R B Cope 36
  • 37. Major Hepatocyte and Cholangiocyte Transporters involved in Bile Formation 01/05/07 Dr R B Cope 37
  • 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 01/05/07 Dr R B Cope 39
  • 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 01/05/07 Dr R B Cope 42
  • 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). 01/05/07 Dr R B Cope 43
  • 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. 01/05/07 Dr R B Cope 44
  • 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. 01/05/07 Dr R B Cope 45
  • 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. 01/05/07 Dr R B Cope 46
  • 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. 01/05/07 Dr R B Cope 47
  • 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. 01/05/07 Dr R B Cope 48
  • 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. 01/05/07 Dr R B Cope 49
  • 50. Globus pallidus staining with bilirubin 01/05/07 Dr R B Cope 50
  • 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. 01/05/07 Dr R B Cope 51
  • 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. 01/05/07 Dr R B Cope 52
  • 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) 01/05/07 Dr R B Cope 53
  • 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! 01/05/07 Dr R B Cope 54
  • 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). 01/05/07 Dr R B Cope 55
  • 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. 01/05/07 Dr R B Cope 56
  • 57. 01/05/07 Dr R B Cope 57
  • 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). 01/05/07 Dr R B Cope 58
  • 59. Ascites following severe liver disease. Note the eversion of the umbilicus. 01/05/07 Dr R B Cope 59
  • 60. Mid-level abdominal CT scans. Left = normal; Right = ascites secondary to liver failure. 01/05/07 Dr R B Cope 60
  • 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). 01/05/07 Dr R B Cope 61
  • 62. Caput medusae associated with portal hypertension, portosystemic shunting and severe liver disease. 01/05/07 Dr R B Cope 62
  • 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). 01/05/07 Dr R B Cope 63
  • 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. 01/05/07 Dr R B Cope 64
  • 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) 01/05/07 Dr R B Cope 65
  • 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.” 01/05/07 Dr R B Cope 66
  • 67. Secondary photosensitization of the face due to 01/05/07 sporodesmin poisoning in a sheep Dr R B Cope 67
  • 68. Severe secondary photosensitzation of the udder of a cow with advanced hepatic disease (again due to sporodesmin) 01/05/07 Dr R B Cope 68
  • 69. Section 2: Responses of the Liver to Toxic Injury. 01/05/07 Dr R B Cope 69
  • 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. 01/05/07 Dr R B Cope 70
  • 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. 01/05/07 Dr R B Cope 71
  • 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. 01/05/07 Dr R B Cope 72
  • 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! 01/05/07 Dr R B Cope 73
  • 74. Hepatocellular Adaptive Responses. • Represent adaptive responses to xenobiotic response rather than hepatocellular damage per se. • Used as histological markers of xenobiotic exposure. 01/05/07 Dr R B Cope 74
  • 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. 01/05/07 Dr R B Cope 75
  • 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. 01/05/07 Dr R B Cope 76
  • 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). 01/05/07 Dr R B Cope 77
  • 78. Centrilobular hepatocyte hypertrophy in a mouse treated with phenobarbital for 8 months. 01/05/07 Dr R B Cope 78
  • 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 01/05/07 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. 01/05/07 Dr R B Cope 80
  • 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 01/05/07 Dr R B Cope 81
  • 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. 01/05/07 82 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. 01/05/07 Dr R B Cope 83
  • 84. Derived from the UK PSD guideline (included as an appendix to the notes) 01/05/07 Dr R B Cope 84
  • 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. 01/05/07 Dr R B Cope 85
  • 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. 01/05/07 Dr R B Cope 86
  • 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. 01/05/07 Dr R B Cope 87
  • 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 01/05/07 Dr R B Cope 89
  • 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
  • 104. Periportal degeneration and portal cirrhosis. 01/05/07 Dr R B Cope 104
  • 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
  • 115. Periportal Bile duct hyperplasia. 01/05/07 Dr R B Cope 115
  • 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
  • 130. Cholestasis 01/05/07 Dr R B Cope 130
  • 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
  • 143. Progression to Neoplasia: Dichloroacetic Acid (DCA) Carcinoma 01/05/07 Dr R B Cope 143
  • 144. 01/05/07 Dr R B Cope 144
  • 145. 01/05/07 Dr R B Cope 145
  • 146. 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
  • 147. Basophilic FHA 01/05/07 Dr R B Cope 147
  • 148. Eosinophilic FHA 01/05/07 Dr R B Cope 148
  • 149. Clear Cell FHA 01/05/07 Dr R B Cope 149
  • 150. Mixed FHA 01/05/07 Dr R B Cope 150
  • 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
  • 155. GST-P Positive FHA 01/05/07 Dr R B Cope 155
  • 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
  • 162. Adenoma Acinar Type (An adenoma is a benign tumor (-oma) of glandular origin) 01/05/07 Dr R B Cope 162
  • 163. 01/05/07 Dr R B Cope 163
  • 164. Adenoma Trabecular Type (An adenoma is a benign tumor (-oma) of glandular origin) 01/05/07 Dr R B Cope 164
  • 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
  • 168. Carcinoma Trabecular Type (Malignant) 01/05/07 Dr R B Cope 168
  • 169. Carcinoma Acinar Type (Malignant) What is this?? 01/05/07 Dr R B Cope 169
  • 170. Carcinoma Clear Cell Type (Malignant) 01/05/07 Dr R B Cope 170
  • 171. Carcinoma Scirrhous Type (Malignant) 01/05/07 Dr R B Cope 171
  • 172. Carcinoma Poorly Differentiated (Malignant) 01/05/07 Dr R B Cope 172
  • 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
  • 183. 01/05/07 Dr R B Cope 183
  • 184. Relevance Depends on MOA 01/05/07 Dr R B Cope 184
  • 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). 01/05/07 Dr R B Cope 195
  • 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. 01/05/07 Dr R B Cope 196
  • 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). 01/05/07 Dr R B Cope 197
  • 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. 01/05/07 Dr R B Cope 198
  • 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 01/05/07 on their apical hepatocyte cell membranes. 199
  • 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. 01/05/07 Dr R B Cope 200
  • 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. 01/05/07 Dr R B Cope 201
  • 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. 01/05/07 Dr R B Cope 202
  • 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. 01/05/07 Dr R B Cope 203
  • 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. 01/05/07 Dr R B Cope 204
  • 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. 01/05/07 Dr R B Cope 205
  • 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. 01/05/07 Dr R B Cope 206
  • 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. 01/05/07 Dr R B Cope 207
  • 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. 01/05/07 Dr R B Cope 208
  • 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. 01/05/07 Dr R B Cope 209
  • 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. 01/05/07 Dr R B Cope 210
  • 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! 01/05/07 Dr R B Cope 211
  • 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. 01/05/07 Dr R B Cope 212
  • 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. 01/05/07 Dr R B Cope 213
  • 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. 01/05/07 Dr R B Cope 214
  • 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. 01/05/07 Dr R B Cope 215