Liver nicnas-nov-2012

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Liver nicnas-nov-2012

  1. 1. An Introduction to the Toxicology of the Liver & Rodent Stomach. Rhian B. Cope BVSc BSc(Hon 1) PhD DABT ERT01/05/07 Dr R B Cope 1
  2. 2. Yes, there is a lot of basic science.It is included deliberately: if you do not understandthe fundamentals of how and why the liver reacts toxenobiotics, you cannot really understand thesignificance and human-relevance of the changesthat occur.Understanding the mode of action is the key to justabout everything in toxicology and toxicological riskassessment.Please bear with me. 01/05/07 Dr R B Cope 2
  3. 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 ClassicalClinical Signs of Hepatic Disease.Section 2: Responses of the Liver to Toxic InjurySection 3: Interpretation of Rodent Hepatic Tumour Data: The Human-Relevance FrameworkSection 4: Detection/ Measurement/Assessment of Hepatic Toxicity.Section 5: The Two Basic Classes of Hepatic Toxicants, and Classical “MustKnow” 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. 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. 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. 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. 7. Structure of the Liver Lobule. Low magnification view ofBthe a liver lobule in the pig01/05/07 Dr R Cope 7
  8. 8. Structure of the Liver Lobule.01/05/07 Low magnification view B Cope human liver lobule Dr R of the 8
  9. 9. Structure of the Liver Lobule.01/05/07 Dr R B Cope 9
  10. 10. Structure of the Liver Lobule.01/05/07 Dr R B Cope 10
  11. 11. Structure of the Liver Lobule.Note the lack of anendothelial basementmembrane, largeendothelial pores andlarge endocytic vacuoles.What are the keytoxicologicalconsequences of thesefeatures? 01/05/07 Dr R B Cope 11
  12. 12. Structure of the Liver Acinus.01/05/07 Dr R B Cope 12
  13. 13. Structure of the Liver Acinus.01/05/07 Dr R B Cope 13
  14. 14. Structure of the Liver Acinus.01/05/07 Dr R B Cope 14
  15. 15. Structure of the Liver Acinus.01/05/07 Dr R B Cope 15
  16. 16. 01/05/07 Dr R B Cope 16
  17. 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. 18. Describe the distribution of damage (necrosis) in this liver01/05/07 Dr R B Cope 18 section using the “lobular” and “acinar” system.
  19. 19. ? Describe the distributionDr R B Cope01/05/07 of damage (necrosis) in this liver 19 section using the “lobular” and “acinar” system.
  20. 20. Central Vein Describe the distributionDr R B Cope01/05/07 of damage (necrosis) in this liver 20 section using the “lobular” and “acinar” system.
  21. 21. Central Vein01/05/07 Centrilobular orB Zone 3 Necrosis. Dr R Cope 21
  22. 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. 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. 24. Hepatocyte Zonal Specialization. Parameter Zone 1 Zone 2 Zone 3 Oxygen tension and level High Intermediate Lowof 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 metabolismCYP level (particularly Cyp Lower Intermediate High 2E1) Level of fatty acid High Intermediate Lowoxidation, gluconeogeneis, and ureagenesisConcentration 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. 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. 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. 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. 28. Pigment accumulation within Kupffer cells.01/05/07 Dr R B Cope 28
  29. 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. 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. 31. Congestive cirrhosis (replacement of hepatocytes with fibrous tissue)secondary to right sided heart failure, trichrome stain. Remember: Ito cellsare responsible for the laying down of new collagen within the liver. WHATARE THE CRITICAL FUNCTIONAL CONSEQUENCES OF SUCH AREACTION IN THE LIVER? 01/05/07 Dr R B Cope 31
  32. 32. A Concise Summary of Key Hepatic Functions01/05/07 32
  33. 33. Consequences of Disruption of Hepatic Function Consequences 3301/05/07
  34. 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 compounds01/05/07 Dr R B Cope 34
  35. 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. 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. 37. Major Hepatocyte and Cholangiocyte Transporters involved in Bile Formation01/05/07 Dr R B Cope 37
  38. 38. Major Hepatocyte Involved in Bile FormationBasolateral Transporters FunctionNa+-taurocholate-co-transporting Uptake of conjugated bile acids,peptide (NTCP) estrogensOrganic 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, digoxinOrganic cation transporter I (OCT I) Divalent lipophilic cations, xenobiotics that contain a tertiary or quarternary amine groupBilitranslocase Bilirubin, sulfobromophthalein; inhibited by phenylmethyl-sulphonyl fluoride; exists in two metastable forms: high and low affinity.
  39. 39. Major Hepatocyte Transporters Involved in Bile Formation.BasolateralTransporters FunctionOrganic 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. 40. Major Hepatocyte Transporters Involved in Bile Formation.Apical Transporters FunctionMultidrug 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 cationicwhich 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, sulfateproteins (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. 41. Hepatic Bilirubin Excretion.Heme containing proteins (Hb,Mb, CYP450) Hepatocyte Sinusoid Reticuloendothelial system Alb Bile canaliculus Spleen, Kupffer cells,Free heme (red) UDP-glucuronideHeme OATP *oxygenase Br BrBiliverdin (green)Biliverdin Alb-Br BT *reductase UGT-1A1Bilirubin (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. 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 kidneys01/05/07 Dr R B Cope 42
  43. 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. 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. 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. 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. 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. 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. 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. 50. Globus pallidus staining with bilirubin01/05/07 Dr R B Cope 50
  51. 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. 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. 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. 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. 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. 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. 57. 01/05/07 Dr R B Cope 57
  58. 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. 59. Ascites following severe liver disease. Note the eversion of the umbilicus.01/05/07 Dr R B Cope 59
  60. 60. Mid-level abdominal CT scans. Left = normal; Right = ascites secondary to liver failure. 01/05/07 Dr R B Cope 60
  61. 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 assumedposture due to intermittent sustained contraction of muscle groups;characteristic of hepatic coma; often assessed by asking the patient to write ordraw simple pictures (e.g. draw a clock face).01/05/07 Dr R B Cope 61
  62. 62. Caput medusae associated with portal hypertension, portosystemic shunting and severe liver disease.01/05/07 Dr R B Cope 62
  63. 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. 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. 65. Clinical Signs of Advanced Hepatocellular or Cholestatic Disease in Ruminants: Secondary Photosensization. In ruminants: Rumen bacteriaChlorophyll 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. 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. 67. Secondary photosensitization of the face due to01/05/07 sporodesmin poisoning in a sheep Dr R B Cope 67
  68. 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. 69. Section 2: Responses of the Liver to Toxic Injury.01/05/07 Dr R B Cope 69
  70. 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. 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. 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. 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. 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. 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. 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. 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. 78. Centrilobular hepatocyte hypertrophy in a mouse treated with phenobarbital for 8 months.01/05/07 Dr R B Cope 78
  79. 79. Centrilobular hepatocyte hypertrophy in a mouse treated withphenobarbital 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. 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. 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. 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. 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. 84. Derived from the UK PSD guideline (included as an appendix to the notes)01/05/07 Dr R B Cope 84
  85. 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. 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. 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
  88. 88. Hepatocyte hydropic degeneration.01/05/07 Dr R B Cope 88
  89. 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 hepatocytes01/05/07 Dr R B Cope 89
  90. 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. 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. 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. 93. Human liver. Fatty change due to alcohol. Note the color. Surface will feel “greasy”.01/05/07 93
  94. 94. Hepatocyte fatty change due to ethanol exposure. Note: fat droplets appear clear due to their extraction during tissue01/05/07 processing. 94
  95. 95. Fine needle aspirates of hepatocytes. Normal on01/05/07 the left, fatty change on the right. 95
  96. 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. 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. 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. 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 for01/05/07 hepatocellular injury. 99
  100. 100. Centrilobular necrosis.01/05/07 Dr R B Cope 100
  101. 101. Hepatic centrilobular necrosis.01/05/07 Dr R B Cope 101
  102. 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. 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. 104. Periportal degeneration and portal cirrhosis.01/05/07 Dr R B Cope 104
  105. 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. 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. 107. Hepatic massive necrosis. Note the periportal accumulation of bile pigments.01/05/07 Dr R B Cope 107
  108. 108. Hepatic massive necrosis.01/05/07 Dr R B Cope 108
  109. 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. 110. Nodular regeneration and periportal cirrhosis following massive necrosis. Trichrome stain. Note that the regenerating liver nodules vary in size and are highlydisorganized. 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. 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. 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. 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. 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. 115. Periportal Bile duct hyperplasia.01/05/07 Dr R B Cope 115
  116. 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. 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. 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 rodents01/05/07 Dr R B Cope 118
  119. 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. 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. 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 Schmorls 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. 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. 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. 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. 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. 126. Oval cell hyperplasia in a mouse exposed to a hepatic carcinogen.01/05/07 Dr R B Cope 126
  127. 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. 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. 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. 130. Cholestasis01/05/07 Dr R B Cope 130
  131. 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. 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. 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. 134. Gross morphology of human liver showing evidence of cholestasis: note the color.01/05/07 Dr R B Cope 134
  135. 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. 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. 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. 138. Section 3: Rodent Liver Tumours and Human Health Risk Assessment01/05/07 Dr R B Cope 138
  139. 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. 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. 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. 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. 143. Progression to Neoplasia: Dichloroacetic Acid (DCA) Carcinoma01/05/07 Dr R B Cope 143
  144. 144. 01/05/07 Dr R B Cope 144
  145. 145. 01/05/07 Dr R B Cope 145
  146. 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. 147. Basophilic FHA01/05/07 Dr R B Cope 147
  148. 148. Eosinophilic FHA01/05/07 Dr R B Cope 148
  149. 149. Clear Cell FHA01/05/07 Dr R B Cope 149
  150. 150. Mixed FHA01/05/07 Dr R B Cope 150
  151. 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. 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. 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 TSG01/05/07 Dr R B Cope 153
  154. 154. Foci of Hepatocellular Alteration: “Pre-neoplastic” change • Mutations of ß-catenin • Decreased apoptosis; • Clonal origin demonstrable in vitro01/05/07 Dr R B Cope 154
  155. 155. GST-P Positive FHA01/05/07 Dr R B Cope 155
  156. 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. 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. 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. 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. 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
  161. 161. Focal hepatocyte adenoma01/05/07 Dr R B Cope 161
  162. 162. Adenoma Acinar Type(An adenoma is a benign tumor (-oma) of glandular origin) 01/05/07 Dr R B Cope 162
  163. 163. 01/05/07 Dr R B Cope 163
  164. 164. Adenoma Trabecular Type(An adenoma is a benign tumor (-oma) of glandular origin) 01/05/07 Dr R B Cope 164
  165. 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. 166. Adenoma – Human Vs Rodent • Humans • Difficult to differentiate from regenerative hyperplasia • Usually solitary • Usually encapsulated01/05/07 Dr R B Cope 166
  167. 167. CarcinomaCarcinoma: Carcinoma refers to an invasive malignant tumorconsisting of transformed epithelial cells. Alternatively, it refers toa malignant tumor composed of transformed cells of unknownhistogenesis, but which possess specific molecular or histologicalcharacteristics that are associated with epithelial cells, such as theproduction of cytokeratins or intercellular bridges. 01/05/07 Dr R B Cope 167
  168. 168. Carcinoma Trabecular Type (Malignant) 01/05/07 Dr R B Cope 168
  169. 169. Carcinoma Acinar Type (Malignant) What is this?? 01/05/07 Dr R B Cope 169
  170. 170. Carcinoma Clear Cell Type (Malignant) 01/05/07 Dr R B Cope 170
  171. 171. Carcinoma Scirrhous Type (Malignant) 01/05/07 Dr R B Cope 171
  172. 172. Carcinoma Poorly Differentiated (Malignant) 01/05/07 Dr R B Cope 172
  173. 173. What is so important about this? 01/05/07 Dr R B Cope 173
  174. 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. 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. 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. 177. So what sort of tumor is this?01/05/07 Dr R B Cope 177
  178. 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. 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. 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. 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. 182. • Observation of tumours under different circumstances lendssupport to the significance of the findings for animalcarcinogenicity. Significance is generally increased by theobservation 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. 183. 01/05/07 Dr R B Cope 183
  184. 184. Relevance Depends on MOA 01/05/07 Dr R B Cope 184
  185. 185. Section 4: Detection and Measurement of Liver Injury01/05/07 Dr R B Cope 185
  186. 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. 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. 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. 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. 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 treatment01/05/07 Dr R B Cope 190
  191. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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

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