1. Hepatic Toxicity UNC School of Public Health
Friday - Nov. 3 Introduction to the liver
Gary Boorman, NIEHS
Monday - Nov. 6 Molecular aspects of liver injury Dr. Robert Sills, NIEHS
Wednesday - Nov. 8 Biochemistry of hepatic injury
Dr. LeCluyse, CellzDirect, Inc.
Friday Nov. 10 Acute Responses to Hepatic Injury
Dr. Rich Miller GSK
Monday, Nov 13 Chronic effects of hepatic injury
Dr. Amy Brix, EPL

2. Why is the Liver so Important in Toxicology?
Hepatotoxicity is the major reason for rejecting new drugs in clinical trials
and withdrawal of drugs already in use
Major metabolic organ
Hepatotoxicity is quite common
Cirrhosis - One of top ten causes of death
Model for cancer mechanisms

3. Liver in Toxicogenomics
The liver is currently the major tissue for Toxicogenomics
> 6,200 references on gene expression and liver in last three years
Easily accessible large parenchymal organ
Model for defining toxic responses
Gene changes reflect systemic responses

4. Lecture Outline Role of the liver Anatomy of the liver
Organization of the liver
Cells of the liver
Rodent diseases affecting liver

5. Liver is Complex Organ Largest organ in body 25% of cardiac output
1500 g in humans (2% of BW)
15 g in male rat (4% of BW)
1.5 g in male mouse (6% of BW)
25% of cardiac output
2 L/Minute in 70 kg Human
Source of most plasma proteins
Interface between food and energy needs
Largest source of fixed macrophages
Marked circadian rhythm

6. Role of the Liver Processes, dietary proteins, carbohydrates, lipids
Stores and releases energy
Exports Glucose from glycogen
Exports Acetoacetate from fatty acids
Detoxification
Endogenous & Exogenous compounds
Oxidation and reduction
Conjugation and hydrolysis
Important role in vitamins
Active synthesis of some forms of B vitamins
Proteins for transport of vitamins
Retinoid storage and metabolism

7. Role of the Liver Acute Phase Response Phagocytosis of particulates
Transient increase or decrease in plasma proteins
Systemic response to local injury
Phagocytosis of particulates
Critical location with blood flow from GI tract
Central role in cholesterol homeostasis
Critical for iron, zinc and copper metabolism

8. Maronpot, Pathology of the mouse. 1999
Question: Is there evidence of a carcinogenic effect due to this compound?
Points for discussion:
Is it okay to combine HCAd and HCCas? Is it necessary?
Is there a shift (progression) to malignancy? If so, does that constitute a carcinogenic effect?
Does it matter if it’s only liver tumors in mice?
Ancillary information:
**p<0.01 with Fisher’s exact t-test
50 animals per group, number of animals with the lesion are listed in the box
2-year exposure
This compound is/is not genotoxic
There was/was not an increase in eosinophilic foci, other foci
Gavage exposure, Similar survival rates, Similar body weight gains
No effect seen in other species (rat), or other gender (female)
No real difference of opinion between pathologists with regards to diagnoses
Maronpot, Pathology of the mouse. 1999

9. Maronpot, Pathology of the mouse. 1999
Question: Is there evidence of a carcinogenic effect due to this compound?
Points for discussion:
Is it okay to combine HCAd and HCCas? Is it necessary?
Is there a shift (progression) to malignancy? If so, does that constitute a carcinogenic effect?
Does it matter if it’s only liver tumors in mice?
Ancillary information:
**p<0.01 with Fisher’s exact t-test
50 animals per group, number of animals with the lesion are listed in the box
2-year exposure
This compound is/is not genotoxic
There was/was not an increase in eosinophilic foci, other foci
Gavage exposure, Similar survival rates, Similar body weight gains
No effect seen in other species (rat), or other gender (female)
No real difference of opinion between pathologists with regards to diagnoses
Maronpot, Pathology of the mouse. 1999

10. Lobe Differences with Acetaminophen
50, 150, 1500, and 2000 mg/kg
For each rat evaluated both
left (L) and median (M) lobe
Yellow highlights dose
Pink highlights Left Lobe

11. Acute Phase Proteins show remarkable Lobe dichotomy
Ttr = Transthyretin
Fgb = Fibrinogen, beta
Ahsg = alpha-2-HS glycoprotein
Tf = transferrin
Significantly DOWN regulated Median (M) lobe
Significantly UP regulated Left (L) lobe
WHY?

12. Lobe variability Gene expression Copper distribution (R > L)
Cancer (R > L)
Cirrhosis of right lobe
Hypertrophy of left lobe
Cirrhosis of left lobe
No Hypertrophy of right lobe
Jim Swenberg is on an article showing more cancer in the right lobe versus the left

13. Why are there lobe differences?
Umbilical blood flows to left lobe (Fetus)
Right and median lobes receive portal blood
Left better oxygenated during development
Left more directly exposed to maternal toxins
Differential portal flow to lobes (Adult)
Blood from stomach to left lobe
Blood flow from colon to right lobe
Colonic cancer metastatic more to right lobe
Blood flow from spleen to left lobe
Contains hepatic growth factors
May explain cirrhosis and hypertrophy

14. Organization of Liver Hepatic plates with bile canicular system
Dual blood supply
75% portal vein (low in oxygen)
25% hepatic artery (high in oxygen)
Blood collects in hepatic vein

15. Question: Is there evidence of a carcinogenic effect due to this compound?
Points for discussion:
Is it okay to combine HCAd and HCCas? Is it necessary?
Is there a shift (progression) to malignancy? If so, does that constitute a carcinogenic effect?
Does it matter if it’s only liver tumors in mice?
Ancillary information:
**p<0.01 with Fisher’s exact t-test
50 animals per group, number of animals with the lesion are listed in the box
2-year exposure
This compound is/is not genotoxic
There was/was not an increase in eosinophilic foci, other foci
Gavage exposure, Similar survival rates, Similar body weight gains
No effect seen in other species (rat), or other gender (female)
No real difference of opinion between pathologists with regards to diagnoses
Tissues and Organs:a text of scanning electron microscopy, Kessel, RG and Kardon,RH, 1979

16. Approximately 1 million classic lobules per liver
Question: Is there evidence of a carcinogenic effect due to this compound?
Points for discussion:
Is it okay to combine HCAd and HCCas? Is it necessary?
Is there a shift (progression) to malignancy? If so, does that constitute a carcinogenic effect?
Does it matter if it’s only liver tumors in mice?
Ancillary information:
**p<0.01 with Fisher’s exact t-test
50 animals per group, number of animals with the lesion are listed in the box
2-year exposure
This compound is/is not genotoxic
There was/was not an increase in eosinophilic foci, other foci
Gavage exposure, Similar survival rates, Similar body weight gains
No effect seen in other species (rat), or other gender (female)
No real difference of opinion between pathologists with regards to diagnoses

17. Rappaport Unit Zone 1 (central or periportal) Largest hepatocytes
More mitochondria
More granular ER
Glycogen metabolism
Glucuronidation of xenobiotics
Formation of plasma proteins
Best oxygenated
Highest concentration of bile salts
More Kupffer cells

18. Portal “triad” (zone 1) hepatic artery (1-2) portal vein (1)
bile duct (1-2)
lymphatics
nerves
connective tissue-collagen type I
Question: Is there evidence of a carcinogenic effect due to this compound?
Points for discussion:
Is it okay to combine HCAd and HCCas? Is it necessary?
Is there a shift (progression) to malignancy? If so, does that constitute a carcinogenic effect?
Does it matter if it’s only liver tumors in mice?
Ancillary information:
**p<0.01 with Fisher’s exact t-test
50 animals per group, number of animals with the lesion are listed in the box
2-year exposure
This compound is/is not genotoxic
There was/was not an increase in eosinophilic foci, other foci
Gavage exposure, Similar survival rates, Similar body weight gains
No effect seen in other species (rat), or other gender (female)
No real difference of opinion between pathologists with regards to diagnoses
Pathology of the Liver, MacSween RNM et al, 2002

19. Glucose-6-phosphatase
Question: Is there evidence of a carcinogenic effect due to this compound?
Points for discussion:
Is it okay to combine HCAd and HCCas? Is it necessary?
Is there a shift (progression) to malignancy? If so, does that constitute a carcinogenic effect?
Does it matter if it’s only liver tumors in mice?
Ancillary information:
**p<0.01 with Fisher’s exact t-test
50 animals per group, number of animals with the lesion are listed in the box
2-year exposure
This compound is/is not genotoxic
There was/was not an increase in eosinophilic foci, other foci
Gavage exposure, Similar survival rates, Similar body weight gains
No effect seen in other species (rat), or other gender (female)
No real difference of opinion between pathologists with regards to diagnoses
Teutsch, et al Hepatology 29:

20. Allyl Alcohol-Induced Necrosis in Zone 1PP
THV
THV PP

21. Rappaport Unit
Zone 3 (More peripheral - Terminal Hepatic Venule) (most commonly Centrilobular)
Smaller hepatocytes
Fewer mitochondria
More agranular ER
Fat and pigment storage
Reductase reactions
Enzyme induction may occur
More susceptible to anoxia

22. Acetaminophen Necrosis in Zone 31 2 3
THV

23. Sometimes lesions are patchy involving more than a lobule
3327

24. Rat caudate lobe with resin in the portal vein demonstrating three dimensional units
Terminal portal branches are located evenly through out the liver and are positioned parallel to neighboring ones (arrow to arrow, arrowhead to arrowhead of * to *). This is a whole mount of the liver giving almost a 3-D view. “E” notes the edge of the liver. Keep this figure in mind next time homogenizing a small piece of liver for DGE!!
Bhunchet and Wake, Hepatology 27: , , 1998

25. Vascular damage following acetaminophen (MRI image)
Malarkey, Ryan, Johnson, Maronpot, 2004

26. Temporal Aspects are also important in the liver

27. Gene Expression for Metallothionein 1a
varies by time during the 24 hour day
CT times; light : Dark 12-24

28. Phase I enzymes that vary by age in the rat

29. Hepatic CYP expression varies with age of rat
What might this mean for a study where rats are exposed 2 years ?

30. Hepatic dimensions Liver is a heterogeneously complex organ Space
Variation by lobe
Zones within lobes
Three-dimensional parenchymal units
Time
Variation by time of day
Variation by age

31. Heterogeneity of liver
Epithelial Cells
Hepatocytes
Cholangiocyes
Mesenchymal cells
Kupffer cells
Endothelial cells
Stellate cells
Other Hepatic components
Smooth muscle cells (blood vessels)
Mesothelia (capsule)
Nerves (unmyelinated)
Neuroendocrine cells
Hematopoeitic cells
Extracellular matrix
5-10% of liver is collagen

32. Heterogeneity of liver
Hepatocytes (60%)
Biliary epithelium (3-5%)
Endothelia (20%)
sinusoids
blood vessels (arteries and veins)
lymphatics
Kupffer cells (15%)
Hepatic stellate cells (5 -%)
Lymphocytes (Pit cells)

33. 5 major players
Question: Is there evidence of a carcinogenic effect due to this compound?
Points for discussion:
Is it okay to combine HCAd and HCCas? Is it necessary?
Is there a shift (progression) to malignancy? If so, does that constitute a carcinogenic effect?
Does it matter if it’s only liver tumors in mice?
Ancillary information:
**p<0.01 with Fisher’s exact t-test
50 animals per group, number of animals with the lesion are listed in the box
2-year exposure
This compound is/is not genotoxic
There was/was not an increase in eosinophilic foci, other foci
Gavage exposure, Similar survival rates, Similar body weight gains
No effect seen in other species (rat), or other gender (female)
No real difference of opinion between pathologists with regards to diagnoses

34. Hepatocytes 80% of mass; 60% of cell numbers
SER and RER (15% of cell volume)
Free ribosomes
Golgi complex
Lysosomes (~ 30 per cell)
Peroxisomes / microbodies (~ 500 per cell)
Mitochondria (1000 per cell)
Cytoskeleton (microfilaments, intermediate filaments, microtubules)
Glycogen
Produces bile (~ 15 ml per kg per day)
Question: Is there evidence of a carcinogenic effect due to this compound?
Points for discussion:
Is it okay to combine HCAd and HCCas? Is it necessary?
Is there a shift (progression) to malignancy? If so, does that constitute a carcinogenic effect?
Does it matter if it’s only liver tumors in mice?
Ancillary information:
**p<0.01 with Fisher’s exact t-test
50 animals per group, number of animals with the lesion are listed in the box
2-year exposure
This compound is/is not genotoxic
There was/was not an increase in eosinophilic foci, other foci
Gavage exposure, Similar survival rates, Similar body weight gains
No effect seen in other species (rat), or other gender (female)
No real difference of opinion between pathologists with regards to diagnoses

35. Lobular Heterogeneity: The Streaming Liver
Question: Is there evidence of a carcinogenic effect due to this compound?
Points for discussion:
Is it okay to combine HCAd and HCCas? Is it necessary?
Is there a shift (progression) to malignancy? If so, does that constitute a carcinogenic effect?
Does it matter if it’s only liver tumors in mice?
Ancillary information:
**p<0.01 with Fisher’s exact t-test
50 animals per group, number of animals with the lesion are listed in the box
2-year exposure
This compound is/is not genotoxic
There was/was not an increase in eosinophilic foci, other foci
Gavage exposure, Similar survival rates, Similar body weight gains
No effect seen in other species (rat), or other gender (female)
No real difference of opinion between pathologists with regards to diagnoses
Pathology of the Liver, MacSween RNM et al, 2002

36. Gene expression varies along the hepatic plate
What might happen to Glutamine synthetase (GS)
Transcript levels with zone 3 necrosis?

37. Endothelial Cells 20% of liver cells, 3.3% of protein content
Discontinuous individual cells/fenestrated
Sieve plates - clustered fenestrate
Direct access of blood to hepatocytes
Gives rise to vascular tumors
Vinyl chloride hemangiosarcomas - human carcinogen

38. Sinusoidal endothelial cells
Question: Is there evidence of a carcinogenic effect due to this compound?
Points for discussion:
Is it okay to combine HCAd and HCCas? Is it necessary?
Is there a shift (progression) to malignancy? If so, does that constitute a carcinogenic effect?
Does it matter if it’s only liver tumors in mice?
Ancillary information:
**p<0.01 with Fisher’s exact t-test
50 animals per group, number of animals with the lesion are listed in the box
2-year exposure
This compound is/is not genotoxic
There was/was not an increase in eosinophilic foci, other foci
Gavage exposure, Similar survival rates, Similar body weight gains
No effect seen in other species (rat), or other gender (female)
No real difference of opinion between pathologists with regards to diagnoses
Fenestrations
Tissues and Organs: a text of scanning electron microscopy, Kessel, RG and Kardon,RH, 1979

39. Pathology of the Liver, MacSween RNM et al, 2002
Question: Is there evidence of a carcinogenic effect due to this compound?
Points for discussion:
Is it okay to combine HCAd and HCCas? Is it necessary?
Is there a shift (progression) to malignancy? If so, does that constitute a carcinogenic effect?
Does it matter if it’s only liver tumors in mice?
Ancillary information:
**p<0.01 with Fisher’s exact t-test
50 animals per group, number of animals with the lesion are listed in the box
2-year exposure
This compound is/is not genotoxic
There was/was not an increase in eosinophilic foci, other foci
Gavage exposure, Similar survival rates, Similar body weight gains
No effect seen in other species (rat), or other gender (female)
No real difference of opinion between pathologists with regards to diagnoses
Pathology of the Liver, MacSween RNM et al, 2002

40. Kupffer Cells 15% of liver cell population, 2.5% of liver protein
Precursors arise from circulating monocytes
Major component of fixed macrophage system
Ingest particles
May contribute to liver disease
Mediators of inflammation (TNF-alpha)

41. Liver showing hepatocytes (H), Kupffer cells (KC), endothelial cells (EC) and stellate cells (SC)

42. Stellate Cells 5% - 8% of all parenchymal cells
Vitamin A storage and metabolism
Significant source of collagen, hepatic fibrosis
Major player in hepatic regeneration
Control microvascular tone
Ito cell tumors in mice

43. Hepatic Stellate Cells (HSC)
Periportal (PP)
small HSCs
perisinusoidal processes
small volume of lipid droplets
Midzone
elongated
large volume of lipid
intense desmin
Central vein (CV)
elongated & IC processes
vitamin A and desmin reduced

44. Question: Is there evidence of a carcinogenic effect due to this compound?
Points for discussion:
Is it okay to combine HCAd and HCCas? Is it necessary?
Is there a shift (progression) to malignancy? If so, does that constitute a carcinogenic effect?
Does it matter if it’s only liver tumors in mice?
Ancillary information:
**p<0.01 with Fisher’s exact t-test
50 animals per group, number of animals with the lesion are listed in the box
2-year exposure
This compound is/is not genotoxic
There was/was not an increase in eosinophilic foci, other foci
Gavage exposure, Similar survival rates, Similar body weight gains
No effect seen in other species (rat), or other gender (female)
No real difference of opinion between pathologists with regards to diagnoses
Tissues and Organs: a text of scanning electron microscopy, Kessel, RG and Kardon,RH, 1979

45. Hepatic Stellate cells wrap around Endothelial Cells

46. Biliary cells 3% to 5% of liver cell population
Form approximately 2 km of tubules
Tight junctions isolate lumen
Modifies bile
Active in secretion and absorption
Effective communicator with other cells
Contains numerous transporters

47. Prevalence of Rodent Pathogens that affect Liver
Helicobacter Species 31%
Epidemic Diarrhea of Infant Mice
EDIM Virus 15%
Mouse Hepatitis Virus 14%
Rat Parvo Virus 6%
Dr. Lila Riley (University of Missouri Diagnostic Laboratory)

48. Source of Rodent Pathogens
Transfer of transgenic mice
Sharing of biological specimens
Other animals in colony
Feral animals
Animal care personnel & visitors
Dr. Lila Riley (University of Missouri Diagnostic Laboratory)

49. Acute Hepatotoxicity Dr. Sills (This Monday)
Molecular biology of hepatic injury
Dr. Sills will assume that you know hepatic lobules before lecture….
Liver structure is also critical for Dr. Millers lecture the following Friday

50. Thanks to Dave Malarkey for several new slides
Question: Is there evidence of a carcinogenic effect due to this compound?
Points for discussion:
Is it okay to combine HCAd and HCCas? Is it necessary?
Is there a shift (progression) to malignancy? If so, does that constitute a carcinogenic effect?
Does it matter if it’s only liver tumors in mice?
Ancillary information:
**p<0.01 with Fisher’s exact t-test
50 animals per group, number of animals with the lesion are listed in the box
2-year exposure
This compound is/is not genotoxic
There was/was not an increase in eosinophilic foci, other foci
Gavage exposure, Similar survival rates, Similar body weight gains
No effect seen in other species (rat), or other gender (female)
No real difference of opinion between pathologists with regards to diagnoses
Thanks to Dave Malarkey for several new slides