A toxicologic pathology overview of liver toxicity including clinical and anatomic pathology, histopathology of toxic liver lesions, factors that influence liver toxicologic pathology, and case examples for establishing a NOAEL.

1. Hepatotoxicity R. R. Maronpot maronpot@me.com Photomicrographs courtesy of the National Toxicology Program (http://ntp.niehs.nih.gov)
2. Outline • Overview/Classification • Assessment • Clinical signs • Clinical chemistry • Gross pathology • Organ weights • Histopathology • Considerations/Influences/Factors/Tissue repair • Case examples
3. Hepatotoxicity • Predictable hepatotoxins – Acetaminophen – Allyl alcohol – Carbon tetrachloride • Idiosyncratic hepatotoxins – Traglitazone – Bromfenac
4. Ideosyncratic Hepatotoxicity Watkins (2005) Toxicol Pathol 33:1-5.
5. Categories of Hepatotoxicity Histologic Lesion/Type of injury • Degeneration/necrosis/cyto toxicity • Cholestasis • Inflammation Mechanisms • Ca homeostasis disruption • Canicular/cholestatic • Metabolic bioactivation • Autoimmunity • Increased apoptosis • Mitochondrial injury • Non-hepatocyte mediated
6. MECHANISMS OF HEPATOTOXICITY • Definitive (single) etiology versus multi-hit Adverse effects often occur together (domino effect) • hepatocellular degeneration, necrosis • biliary cell degeneration, necrosis • cholestasis • hepatitis (often not a primary finding) • fibrosis, cirrhosis • vascular, sinusoidal effects • pre-neoplastic lesions (altered hepatocellular foci, others), mitogenesis
7. Assessment of Hepatotoxicity • Clinical signs • Clinical chemistry • Gross pathology • Organ weight • Histopathology
8. Clinical Chemistry • Advantages – Serial sampling – Detection of metabolic injury – Detection of organ specific effects – Help establish NOEL – Help determine toxic mechanism • Act within specific cellular localization – Cell membrane – Cytosol – Mitochondria
9. Cellular Localization • Cholephilic analytes & enzymes – Bile acids – Alkaline phosphatase • Cytosolic enzymes – Alanine aminotransferase – Sorbitol dehydrogenase • Membrane enzymes – Gamma glutamyl transpeptidase – Alkaline phosphatase • Mitochondrial enzymes – Glutamate dehydrogenase
10. Recommended Clinical Chemistry for Hepatotoxicity • Hepatocellular toxicity – Alanine aminotransferase (ALT) • Cytosolic • Liver specific • Half-life 48 to 60 hours • Glucocorticoids can increase ALT in rats up to 13X – Sorbitol dehydrogenase (SDH) • Good for hepatocellular injury in all species • Short half-life (<6 hours) • Aspartate aminotransferase (AST) • Lactate dehydrogenase – Total bile acids • Affected by altered enterohepatic circulation and altered hepatic function • Also good indicator of cholestasis
11. Recommended Clinical Chemistry for Hepatotoxicity • Hepatobiliary toxicity – Alkaline phosphatase • Fairly ubiquitous – membranes & brush borders • Bone and placenta • Good marker of cholestasis • Minimal increase in hepatocellular damage – 5’-Nucleotidase • Membrane enzyme • Ubiquitous – kidney and intestine high • Good marker for cholestasis – Total bile acids • Relatively sensitive indicator of cholestasis – Bilirubin, direct and total • Total bilirubin (direct from cholestasis; indirect from hemolytic disease) • Measure total and direct and determine indirect by subtraction
12. Evaluation of Liver Alanine Aminotransferase (ALT, SGPT) – Greatest activity – hepatocytes; also found in skeletal/cardiac muscle – Biological half-life – varies (~48-60 hours) – Can be induced (eg., glucocorticoids – up to 13X increase) – Increased – hepatocellular injury, induction, muscle injury – Decreased – enzyme inhibition (cyclosporin) Sorbitol Dehydrogenase (SDH) – Greatest activity – hepatocytes; also found in testes – Biological half-life – short (≤6 hours) – Sample stability – not as stabile; in rats, stabile refrigerated (~2 days) – Not known to be induced – Only known cause for serum increase – hepatocellular injury or leakage – Good indicator for all species
13. Evaluation of Liver – cont. Aspartate Aminotransferase (AST, SGOT) – Greatest activity – found in numerous tissues (not specific for liver injury) – Biological half-life – short (~15-24 hours) – Red blood cells contain significant amounts (hemolysis – falsely elevates) – Used in past to detect hepatocellular injury (still used for large animals); used for muscle injury Alkaline Phosphatase (ALP) – Greatest activity – liver, bone intestine, kidney, placenta – Biological half-life – isoenzymes of different tissues highly variable – Can be induced (eg., glucocorticoids, phenobarbital, dieldrin) – Increased – cholestasis, drug induction, increased osteoblastic activity, cancer – Decreased – decreased food intake (rats)
14. Evaluation of Liver – cont. Bilirubin, direct (conjugated) and indirect (unconjugated) – Breakdown product of hemoglobin – Direct: conjugation carried out by liver – Increased indirect – Increased hemolysis; decreased hepatic uptake – Increased direct – cholestasis Total Bile Acids (TBA) – Produced by liver – cholic and chenodeoxycholic (primary bile acids) – Taurine or glycine conjugated and secreted into bile – Intestinal bacterial modification produces deoxycholic and lithocholic acids – Increased – cholestasis, decreased hepatic uptake/conjugation, hepatic injury – Decreased – altered enterohepatic recirculation
15. Clinical Chemistry Case Examples ____________________ Ref Value _Case 1 ALT (Alanine aminotransferase) 30-55 IU/L 34 SDH (Sorbitol dehydrogenase) 10-20 IU/L 16 ALP (Alkaline phosphatase) 250-350 IU/L 157 TBA (Total bile acids) 25-35 µmol/L 31 Tbili (Total bilirubin) 0.1-0.5 mg/dL 0.2 Dbili (Direct bilirubin) 0.05-0.2 mg/dL 0.1
16. Clinical Chemistry Case Examples ____________________ Ref Value _Case 1 ALT (Alanine aminotransferase) 30-55 IU/L 34 SDH (Sorbitol dehydrogenase) 10-20 IU/L 16 ALP (Alkaline phosphatase) 250-350 IU/L 157 TBA (Total bile acids) 25-35 µmol/L 31 Tbili (Total bilirubin) 0.1-0.5 mg/dL 0.2 Dbili (Direct bilirubin) 0.05-0.2 mg/dL 0.1
17. Clinical Chemistry Case Examples ____________________ Ref Value _Case 1 ALT (Alanine aminotransferase) 30-55 IU/L 34 SDH (Sorbitol dehydrogenase) 10-20 IU/L 16 ALP (Alkaline phosphatase) 250-350 IU/L 157 TBA (Total bile acids) 25-35 µmol/L 31 Tbili (Total bilirubin) 0.1-0.5 mg/dL 0.2 Dbili (Direct bilirubin) 0.05-0.2 mg/dL 0.1 Decreased ALP – decreased food intake
18. Clinical Chemistry Case Examples ____________________ Ref Value _Case 2 ALT (Alanine aminotransferase) 30-55 IU/L 130 SDH (Sorbitol dehydrogenase) 10-20 IU/L 13 ALP (Alkaline phosphatase) 250-350 IU/L 321 TBA (Total bile acids) 25-35 µmol/L 27 Tbili (Total bilirubin) 0.1-0.5 mg/dL 0.3 Dbili (Direct bilirubin) 0.05-0.2 mg/dL 0.1
19. Clinical Chemistry Case Examples ____________________ Ref Value _Case 2 ALT (Alanine aminotransferase) 30-55 IU/L 130 SDH (Sorbitol dehydrogenase) 10-20 IU/L 13 ALP (Alkaline phosphatase) 250-350 IU/L 321 TBA (Total bile acids) 25-35 µmol/L 27 Tbili (Total bilirubin) 0.1-0.5 mg/dL 0.3 Dbili (Direct bilirubin) 0.05-0.2 mg/dL 0.1
20. Clinical Chemistry Case Examples ____________________ Ref Value _Case 2 ALT (Alanine aminotransferase) 30-55 IU/L 130 SDH (Sorbitol dehydrogenase) 10-20 IU/L 13 ALP (Alkaline phosphatase) 250-350 IU/L 321 TBA (Total bile acids) 25-35 µmol/L 27 Tbili (Total bilirubin) 0.1-0.5 mg/dL 0.3 Dbili (Direct bilirubin) 0.05-0.2 mg/dL 0.1 Increased ALT – suspect enzyme induction
21. Clinical Chemistry Case Examples ____________________ Ref Value _Case 3 ALT (Alanine aminotransferase) 30-55 IU/L 450 SDH (Sorbitol dehydrogenase) 10-20 IU/L 63 ALP (Alkaline phosphatase) 250-350 IU/L 279 TBA (Total bile acids) 25-35 µmol/L 43 Tbili (Total bilirubin) 0.1-0.5 mg/dL 0.3 Dbili (Direct bilirubin) 0.05-0.2 mg/dL 0.1
22. Clinical Chemistry Case Examples ____________________ Ref Value _Case 3 ALT (Alanine aminotransferase) 30-55 IU/L 450 SDH (Sorbitol dehydrogenase) 10-20 IU/L 63 ALP (Alkaline phosphatase) 250-350 IU/L 279 TBA (Total bile acids) 25-35 µmol/L 43 Tbili (Total bilirubin) 0.1-0.5 mg/dL 0.3 Dbili (Direct bilirubin) 0.05-0.2 mg/dL 0.1
23. Clinical Chemistry Case Examples ____________________ Ref Value _Case 3 ALT (Alanine aminotransferase) 30-55 IU/L 450 SDH (Sorbitol dehydrogenase) 10-20 IU/L 63 ALP (Alkaline phosphatase) 250-350 IU/L 279 TBA (Total bile acids) 25-35 µmol/L 43 Tbili (Total bilirubin) 0.1-0.5 mg/dL 0.3 Dbili (Direct bilirubin) 0.05-0.2 mg/dL 0.1 Increased ALT, SDH, TBA – suspect hepatocellular injury
24. Clinical Chemistry Case Examples ____________________ Ref Value _Case 4 ALT (Alanine aminotransferase) 30-55 IU/L 87 SDH (Sorbitol dehydrogenase) 10-20 IU/L 28 ALP (Alkaline phosphatase) 250-350 IU/L 987 TBA (Total bile acids) 25-35 µmol/L 104 Tbili (Total bilirubin) 0.1-0.5 mg/dL 4.7 Dbili (Direct bilirubin) 0.05-0.2 mg/dL 3.1
25. Clinical Chemistry Case Examples ____________________ Ref Value _Case 4 ALT (Alanine aminotransferase) 30-55 IU/L 87 SDH (Sorbitol dehydrogenase) 10-20 IU/L 28 ALP (Alkaline phosphatase) 250-350 IU/L 987 TBA (Total bile acids) 25-35 µmol/L 104 Tbili (Total bilirubin) 0.1-0.5 mg/dL 4.7 Dbili (Direct bilirubin) 0.05-0.2 mg/dL 3.1
26. Clinical Chemistry Case Examples ____________________ Ref Value _Case 4 ALT (Alanine aminotransferase) 30-55 IU/L 87 SDH (Sorbitol dehydrogenase) 10-20 IU/L 28 ALP (Alkaline phosphatase) 250-350 IU/L 987 TBA (Total bile acids) 25-35 µmol/L 104 Tbili (Total bilirubin) 0.1-0.5 mg/dL 4.7 Dbili (Direct bilirubin) 0.05-0.2 mg/dL 3.1 Increased ALT, SDH, ALP, TBA, T & Dbili – suspect biliary obstruction
27. Interpreting Clinical Chemistry • Know the reference range • Know the sampling interval • Enzyme induction versus cellular damage • Magnitude of the change • Alteration of single versus multiple analytes • Correlation with other changes – Clinical signs – Organ weights – Histopathology
28. Outline • Overview/Classification • Assessment • Clinical signs • Clinical chemistry • Gross pathology • Organ weights • Histopathology • Considerations/Influences/Factors/Tissue repair • Case examples
29. Hepatomegaly
30. Dose Male Female (mg/kg) rat rat 0 10.1±0.5 5.6±0.5 0.01 11.2±1.0 5.9±0.3 5 12.3±1.4* 6.5±0.4* 50 16.0±1.6* 8.7±0.5* 100 17.4±1.4* 9.8±0.8* 500 20.0±1.8* 12.2±1.1* * p<0.05. Liver weights (g) in rats treated with flame retardant containing polybrominated diphenyl ethers
31. Outline • Overview/Classification • Assessment • Clinical signs • Clinical chemistry • Gross pathology • Organ weights • Histopathology • Considerations/Influences/Factors/Tissue repair • Case examples
32. Cellular degeneration • Glycogen depletion • Fatty change • Phospholipidosis • Amyloidosis • Mineralization • Pigment deposition • Crystals • Inclusion bodies • Hypertrophy, hepatocellular • Atrophy, hepatocellular
33. Functional Structure of Hepatic Parenchyma
34. Classic lobule
35. >Hepatocytes (80% of parenchyma) >Biliary epithelium >Endothelia > sinusoids > blood vessels (arteries and veins) > lymphatics >Kupffer cells >Hepatic stellate cells >Lymphocytes (Pit cells) Heterogeneity of Liver
36. 5 major players
37. Hepatic stellate cell (5-8% of liver cells) Tissues and Organs: a text of scanning electron microscopy, Kessel, RG and Kardon,RH, 1979
38. >Progenitor cells >Oval cell – rodent models >Hepatoblasts – humans Fibroblasts >Smooth muscle cells (blood vessels) >Mesothelia >Nerves (unmyelinated) >Neuroendocrine cells >Hematopoeitic cells >Blood >Extracellular matrix > 5-10% of liver is collagen Heterogeneity of Liver
39. Histologic Liver Responses • Cytoplasmic alteration – Glycogen deposition and depletion – Fatty change – Pigmentation – Degeneration – Cell death • Apoptosis • Necrosis – Hypertrophy – Karyomegaly – Atrophy • Inflammatory cell infiltrates • Angiectasis • Proliferative responses – Non-neoplastic – Neoplastic • Biliary cysts • Phospholipidosis • Amyloidosis • Crystals • Inclusion bodies
40. Normal Rodent Liver – Fasted Animal
41. Glycogen accumulation
42. Intracytoplasmic GlycogenLiver without Glycogen
43. PAS with diastasePAS
44. N M G L L G M M P RER Glycogen
45. PV CV
46. Normal glycogen accumulation Glycogen depletion Glycogen
47. Normal glycogen accumulation Excessive glycogen Glycogen
48. Histologic Liver Responses • Cytoplasmic alteration – Glycogen deposition and depletion – Fatty change – Pigmentation – Degeneration – Cell death • Apoptosis • Necrosis – Hypertrophy – Karyomegaly – Atrophy • Inflammatory cell infiltrates • Angiectasis • Proliferative responses – Non-neoplastic – Neoplastic • Biliary cysts • Phospholipidosis • Amyloidosis • Crystals • Inclusion bodies
49. Macrovesicular Fatty Change
50. Fatty change and glycogen accumulation
51. Microvesicular Fatty Change
52. Macrovesicular fatty change Microvesicular fatty change
53. Oil-red-O stain for lipid Mouse Liver
54. Glycogen Microvesicular Fat Cytoplasmic Alteration
55. Glycogen and Fatty Change • Glycogen common & often not diagnosed – Treatment effects not common • Fatty change reflects a treatment effect – Typically reversible – Macrovesicular – altered lipid metabolism – Mirovesicular – possible mitochondrial effect • Combinations of glycogen and fatty change
56. Histologic Liver Responses • Cytoplasmic alteration – Glycogen depletion – Fatty change – Pigmentation – Degeneration – Cell death • Apoptosis • Necrosis – Hypertrophy – Karyomegaly – Atrophy • Inflammatory cell infiltrates • Angiectasis • Proliferative responses – Non-neoplastic – Neoplastic • Biliary cysts • Phospholipidosis • Amyloidosis • Crystals • Inclusion bodies
57. Hepatic Pigments • In hepatocytes, canaliculi, bile ductules, bile ducts, Kupffer cells –Bile (cholestasis) –Lipofuscin –Hemoglobin, methemoglobin –Thoratrast / other drugs
58. Pigment Deposition
59. Pigment Deposition
60. Cholestasis
61. Cholestasis (bile) cytoplasmic cananicular
62. Detection of bile Hall’s stain for bile
63. Histologic Liver Responses • Cytoplasmic alteration – Glycogen – Fatty change – Pigmentation – Degeneration – Cell death • Apoptosis • Necrosis – Hypertrophy – Karyomegaly – Atrophy • Inflammatory cell infiltrates • Angiectasis • Proliferative responses – Non-neoplastic – Neoplastic • Biliary cysts • Phospholipidosis • Amyloidosis • Crystals • Inclusion bodies
64. Degeneration and Fatty Change in a Mouse Given Benzene Hexachloride
65. Hydropic Degeneration
66. Cystic Degeneration (Spongiosis hepatis)
67. Pigmentation & Degeneration • In general, pigmentation and degeneration are treatment-related – Cystic degeneration is usually not treatment- related • Severity of change is important • Both are reversible but pigment may take a long time to be removed
68. Histologic Liver Responses • Cytoplasmic alteration – Glycogen depletion – Fatty change – Pigmentation – Degeneration – Cell death • Apoptosis • Necrosis – Hypertrophy – Karyomegaly – Atrophy • Inflammatory cell infiltrates • Angiectasis • Proliferative responses – Non-neoplastic – Neoplastic • Biliary cysts • Phospholipidosis • Amyloidosis • Crystals • Inclusion bodies
69. Apoptosis
70. Apoptosis in a Liver with Increased Peroxisomes in Hepatocytes
71. Single Cell Necrosis or Apoptosis?
72. Necrosis (Oncosis)
73. Necrosis (Oncosis)Apoptosis
74. PERIPORTAL MID-LOBULAR CENTRILOBULAR
75. Centrilobular Hepatocyte Necrosis
76. Centrilobular Hepatocyte Necrosis
77. Centrilobular Hepatocyte Necrosis
78. Centrilobular Hepatocyte Necrosis
79. Bridging Centrilobular Hepatocyte Necrosis
80. Centrilobular necrosis CENTRILOBULAR PORTAL
81. Centrilobular Necrosis with Hemorrhage and Mineralization
82. Centrilobular necrosis & inflammation
83. Midlobular necrosis CV CV
84. Acetaminophen – lobe variation in necrosis
85. 3-D reconstruction Teutsch, et al. 1999. Hepatology 29:494-505
86. Teutsch, et al. 1999. Hepatology 29:494-505
87. Teutsch, et al. 1999. Hepatology 29:494-505
88. Teutsch, et al. 1999. Hepatology 29:494-505
89. MRI study – acetaminophen Control Treated
90. MRI study – acetaminophen Control Treated Hepatic vein Portal vein
91. Necrosis & Apoptosis
92. Outline • Overview/Classification • Assessment • Clinical signs • Clinical chemistry • Gross pathology • Organ weights • Histopathology • Considerations/Influences/Factors/Tissue repair • Case examples
93. Histologic Liver Responses • Cytoplasmic alteration – Glycogen depletion – Fatty change – Pigmentation – Degeneration – Cell death • Apoptosis • Necrosis – Hypertrophy – Karyomegaly – Atrophy • Inflammatory cell infiltrates • Angiectasis • Proliferative responses – Non-neoplastic – Neoplastic • Biliary cysts • Phospholipidosis • Amyloidosis • Crystals • Inclusion bodies
94. Histologic Liver Responses • Cytoplasmic alteration – Glycogen depletion – Fatty change – Pigmentation – Degeneration – Cell death • Apoptosis • Necrosis – Hypertrophy – Karyomegaly – Atrophy • Inflammatory cell infiltrates • Angiectasis • Proliferative responses – Non-neoplastic – Neoplastic • Biliary cysts • Phospholipidosis • Amyloidosis • Crystals • Inclusion bodies
95. Hepatomegaly • Increase cell size (hypertrophy) • Increase cell number (hyperplasia) • Increase blood
96. Dose Male Female (mg/kg) rat rat 0 10.1±0.5 5.6±0.5 0.01 11.2±1.0 5.9±0.3 5 12.3±1.4* 6.5±0.4* 50 16.0±1.6* 8.7±0.5* 100 17.4±1.4* 9.8±0.8* 500 20.0±1.8* 12.2±1.1* * p<0.05. Liver weights (g) in rats treated with flame retardant containing polybrominated diphenyl ethers (PBDEs) Microsomal Enzyme Induction
97. Enzyme Induction
98. Smooth Endoplasmic Reticulum (SER) Proliferation Central Vein
99. N N Smooth Endoplasmic Reticulum (SER) Proliferation Control
100. CYP3A1 Immunohistochemistry Constitutive Induced CV CV Clayton, et al. 2007
101. Common enzyme inducers • CAR (Constitutive Androstane Receptor) • Phenobarbitone & CYP2B • PRX (Pregnane X receptor) • Cyproterone acetate & CYP3A • AhR (Aryl hydrocarbon receptor) • Dioxin & CYP1A1, 1A2, 1B1 • PPAR (Peroxisome Proliferator-Activated Receptor) • Fibrates and Peroxisomes
102. Peroxisome Proliferation
103. Control Treated
104. Peroxisome Proliferation Ultrastructure Control Treated
105. Peroxisome Proliferators • Hypolipidemics – Clofibrate – Gemfibrozil • Methaphenilene • Ibuprofen • Diethylhexyl phthalate
106. Toxicologic significance of enzyme inducers • Increased degradation of hormones • Increased deactivation or activation of xenobiotics • Altered metabolism of drugs
107. Adverse?
108. •Degeneration •Necrosis / apoptosis •Hyperplasia •Hepatocellular •Bile duct •Steatosis / lipidosis •Cholestasis •Karyomegaly When is hepatic enzyme induction adverse?
109. Persistent & Excessive Enzyme Induction
110. Persistent & Excessive Enzyme Induction
111. Karyomegaly
112. Multinucleated Hepatocytes
113. Cholestasis
114. Hepatic Enzyme Induction • Common response to xenobiotic exposure • Considered adaptive and non-adverse in the absence of indications of toxicity – No associated histopathology or markedly abnormal clinical chemistry • May have secondary effects (e.g., thyroid adenomas) • Can be adverse when extreme – Can produce toxicity, can generate oxygen radicals
115. Hypertrophy and Cancer
116. Hypertrophy and Liver Cancer in Mice – enzyme induction > 140% – liver weight >150% at 1 year – induction of p450 enzymes may lead to the formation of oxygen radicals or other electrophilic reactive species capable of causing DNA damage – hyperplasia and degeneration – polyploidy, aneuploidy – high probability of liver cancer at 2 years
117. Histologic Liver Responses • Cytoplasmic alteration – Glycogen depletion – Fatty change – Pigmentation – Degeneration – Cell death • Apoptosis • Necrosis – Hypertrophy – Karyomegaly – Atrophy • Inflammatory cell infiltrates • Angiectasis • Proliferative responses – Non-neoplastic – Neoplastic • Biliary cysts • Phospholipidosis • Amyloidosis • Crystals • Inclusion bodies
118. Inflammation • Acute • Chronic • Chronic active • Granulomatous • Mononuclear cell aggregates • Inflammation, peribiliary • Fibrosis (cirrhosis)
119. Focal Inflammation
120. Focal Necrosis and Inflammation
121. Granulomatous Inflammation
122. Put picture here Extramedullary hematopoesis?
123. Extramedullary hematopoesis?
124. Portal Fibrosis
125. “Cirrhosis” Diallyl phthalate F344 Rat
126. Some Summary Points • Liver has remarkable reserve capacity • Responses can be adaptive – Non-adverse – Adverse • Adverse effects often occur together • Rat liver has a secondary lobular structure that may explain unusual distribution of lesions • Nodular lesions and aggressive proliferative changes in the liver are not necessarily neoplasms
127. Histologic Liver Responses • Cytoplasmic alteration – Glycogen depletion – Fatty change – Pigmentation – Degeneration – Cell death • Apoptosis • Necrosis – Hypertrophy – Karyomegaly – Atrophy • Inflammatory cell infiltrates • Angiectasis • Proliferative responses – Non-neoplastic – Neoplastic • Biliary cysts • Phospholipidosis • Amyloidosis • Crystals • Inclusion bodies
128. Foci of Cellular Alteration
129. Foci of cellular alteration occur spontaneously in older rodents and may be induced by treatment and occur in younger rodents. They are relatively uncommon in young rodents. It is recommended that the occurrence of foci of cellular alteration in prechronic studies be documented and classified into appropriate subtypes.
130. Progression of Proliferative Liver Lesions Basophilic Focus Hepatocellular adenoma Metastatic carcinoma Hepatocellular carcinoma
131. Relationship of Foci and Liver Tumors in F344 Rats • Liver tumor negative studies – No increased incidence of foci at study termination • Liver tumor positive studies – genotoxic agent – 3 to 10-fold increase in multiple foci – Eosinophilic (3-10X); Clear (2-9X); Mixed (3x); Basophilic (4- 8X) • Liver tumor positive studies – non-genotoxic agent – 2 to 15-fold increase in foci – Eosinophilic (4-15X); Mixed (2-5X)
132. Regenerative Hyperplasia; Nodular hyperplasia
133. Oval Cell Proliferation
134. Bile Duct Hyperplasia
135. Outline • Overview/Classification • Assessment • Clinical signs • Clinical chemistry • Gross pathology • Organ weights • Histopathology • Considerations/Influences/Factors/Tissue repair • Case examples
136. •Ability of liver to regenerate is well known •Following injury a cascade of promitogenic signals is triggered •Tissue repair follows a dose response •Tissue repair increases with dose up to a threshold dose •Promitogenic signaling is inhibited by doses above the threshold
137. Chemicals That Induce Tissue Repair • Acetaminophen • Allyl alcohol • Carbon tetrachloride • Choroform • 1,2-Dichlorobenzene • Thioacetamide • Trichloroethylene
138. Considerations in Interpretation of Bioassay Data Neoplasia • Modifying factors • Dose relationships • Trans-sex & trans-species • Common vs. unique lesions • Lesion progression • Species/strain susceptibility • Controls • Lumping & Splitting • Direct vs. indirect causality • Benign vs. malignant • Latency • Multiplicity • Levels of evidence of carcinogenicity Non-neoplasia • Modifying factors • Dose relationships • Trans-sex & trans-species • Common vs. unique lesions • Lesion progression • Species/strain susceptibility • Controls • Lumping & Splitting • Direct vs. indirect causality • Adaptive vs. adverse • Severity • MTD, NOEL and NOAEL
139. Dose and Dose Relationships
140. Figure 1. Serum ALT levels 24 hours after dosing with APAP (300mg/kg) or vehicle (0.5% methylcellulose). From I. Rusyn, University of North Carolina
141. Non-neoplastic Severity Responses Grades of lesions severity used by the NTP: Minimal (1+) Mild (2+) Moderate (3+) Marked (4+) Severe (5+)
142. MTD, NOEL and NOAEL MTD = Maximum tolerated dose NOEL = No observable effect level. Highest dose administered that does not produce toxic effects. NOAEL = No observable adverse effect level. Highest dose administered that does not produce an adverse effect.
143. Case 1 – Chronic hepatic inflammation in a 6-month study. N=20 per group. Is there a real effect here? At what dose?
144. If this is the only change in the study, what is the MTD for purposes of setting a high dose for a 2-year cancer bioassay? Is there a NOAEL?Is there a real effect here? At what dose? Case 1 – Chronic hepatic inflammation in a 6-month study. N=20 per group.
145. Liver necrosis in a 6-month monkey study. N=4 per group. The company wants to market this new anti-inflammatory drug for use in humans. Case 2
146. Liver necrosis in a 6-month monkey study. N=4 per group. The company wants to market this new anti-inflammatory drug for use in humans. Any additional suggestions for your new employer regarding this new drug candidate? What is the NOAEL? You are a new toxicologist just hired by this company. Would you recommend that they take this drug into human clinical trials? What top dose would you recommend that they use? Case 2
147. Case 3 – Liver inflammation in a 6-month rodent toxicity study. Is there a real effect? Is there a good dose response?
148. What is the NOEL? What is the NOAEL? What is the MTD? Case 3 – Liver inflammation in a 6-month rodent toxicity study.
149. Sorting Out the Process Non-neoplastic Putative preneoplastic Neoplastic Exacerbation of background lesions Aging changes Species & strain specificity Xenobiotic specificity Temporal relationships Acute Prechronic Intermediate Chronic Specific hepatic changes often do not occur in isolation.