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.
Hepatotoxicity
View as PDF1. 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