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