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Hepatic Toxicity UNC School of Public Health
Friday - Nov. 3 Introduction to the liverGary 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
Why is the Liver so Important in Toxicology?
Hepatotoxicity is the major reason for rejecting new drugs in clinical trialsand 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
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
Lecture Outline
Role of the liver
Anatomy of the liver
Organization of the liver
Cells of the liver
Rodent diseases affecting liver
Liver is Complex Organ
Largest organ in body1500 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 output2 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
Role of the Liver
Processes, dietary proteins, carbohydrates, lipidsStores and releases energy Exports Glucose from glycogenExports Acetoacetate from fatty acids
Detoxification Endogenous & Exogenous compoundsOxidation and reductionConjugation and hydrolysis
Important role in vitamins Active synthesis of some forms of B vitaminsProteins for transport of vitaminsRetinoid storage and metabolism
Role of the Liver
Acute Phase ResponseTransient increase or decrease in plasma proteins
Systemic response to local injury
Phagocytosis of particulatesCritical location with blood flow from GI tract
Central role in cholesterol homeostasis
Critical for iron, zinc and copper metabolism
Maronpot, Pathology of the mouse. 1999
Maronpot, Pathology of the mouse. 1999
Lobe Differences with Acetaminophen
50, 150, 1500, and 2000 mg/kg
For each rat evaluated both left (L) and median (M) lobe
Yellow highlights dosePink highlights Left Lobe
Acute Phase Proteins show remarkable Lobe dichotomy
Ttr = Transthyretin Fgb = Fibrinogen, betaAhsg = alpha-2-HS glycoproteinTf = transferrin Significantly DOWN regulated Median (M) lobeSignificantly UP regulated Left (L) lobeWHY?
Gene expression
Copper distribution (R > L)
Cancer (R > L)
Cirrhosis of right lobeHypertrophy of left lobe
Cirrhosis of left lobeNo Hypertrophy of right lobe
Lobe variability
Why are there lobe differences?
Umbilical blood flows to left lobe (Fetus)Right and median lobes receive portal bloodLeft better oxygenated during developmentLeft more directly exposed to maternal toxins
Differential portal flow to lobes (Adult)Blood from stomach to left lobeBlood flow from colon to right lobeColonic cancer metastatic more to right lobe
Blood flow from spleen to left lobeContains hepatic growth factorsMay explain cirrhosis and hypertrophy
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
Tissues and Organs:a text of scanning electron microscopy, Kessel, RG and Kardon,RH, 1979
Approximately 1 million classic lobules per liver
Rappaport Unit
Zone 1 (central or periportal)Largest hepatocytesMore mitochondriaMore granular ERGlycogen metabolismGlucuronidation of xenobioticsFormation of plasma proteinsBest oxygenatedHighest concentration of bile saltsMore Kupffer cells
Portal “triad” (zone 1)
hepatic artery (1-2)hepatic artery (1-2)portal vein (1)portal vein (1)bile duct (1-2)bile duct (1-2)lymphaticslymphaticsnervesnerves
connective tissue-collagen type Iconnective tissue-collagen type IPathology of the Liver, MacSween RNM et al, 2002
Teutsch, et al. 1999. Hepatology 29:494-505
Glucose-6-phosphatase
Allyl Alcohol-Induced Necrosis in Zone 1
THV
THV
PP
PP
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
Acetaminophen Necrosis in Zone 3
THV
3 2 1
Sometimes lesions are patchy involving more than a lobule
Bhunchet and Wake, Hepatology 27:481-487, 1998-487, 1998
Rat caudate lobe with resin in the portal vein demonstrating three dimensional units
Vascular damage following acetaminophen (MRI image)
Malarkey, Ryan, Johnson, Maronpot, 2004
Temporal Aspects are also important in the liver
CT times; light 0 - 12: Dark 12-24
Gene Expression for Metallothionein 1avaries by time during the 24 hour day
Phase I enzymes that vary by age in the rat
Hepatic CYP expression varies with age of rat
What might this mean for a study where rats are exposed 2 years ?
Liver is a heterogeneously complex organ
SpaceSpace
Variation by lobe Zones within lobesThree-dimensional parenchymal units
TimeTime
Variation by time of dayVariation by age
Hepatic dimensions
Epithelial CellsHepatocytes Cholangiocyes
Mesenchymal cellsKupffer cellsEndothelial cellsStellate cells
Other Hepatic componentsSmooth muscle cells (blood vessels)Mesothelia (capsule)Nerves (unmyelinated)Neuroendocrine cellsHematopoeitic cellsExtracellular matrix
5-10% of liver is collagen
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)
Heterogeneity of liver
5 major players
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)
Hepatocytes
Lobular Heterogeneity: The Streaming Lobular Heterogeneity: The Streaming LiverLiver
Pathology of the Liver, MacSween RNM et al, 2002
Gene expression varies along the hepatic plate
What might happen to Glutamine synthetase (GS) Transcript levels with zone 3 necrosis?
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
Tissues and Organs: a text of scanning electron microscopy, Kessel, RG and Kardon,RH, 1979
Sinusoidal endothelial cells
Fenestrations
Pathology of the Liver, MacSween RNM et al, 2002
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)
Liver showing hepatocytes (H), Kupffer cells (KC), endothelial cells (EC) and stellate cells (SC)
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
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
Hepatic Stellate Cells (HSC)
Tissues and Organs: a text of scanning electron microscopy, Kessel, RG and Kardon,RH, 1979
Hepatic Stellate cells wrap around Endothelial Cells
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
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)
Source of Rodent Pathogens
Transfer of transgenic mice
Sharing of biological specimens
Other animals in colony
Feral animals
Animal care personnel & visitorsDr. Lila Riley (University of Missouri Diagnostic
Laboratory)
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
Thanks to Dave Malarkey for several new slides