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

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

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 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

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

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

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

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

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

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?

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

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

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

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

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

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

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

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. 1999. Hepatology 29:494-505

Allyl Alcohol-Induced Necrosis in Zone 1 PP THV THV 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 1 2 3 THV

Sometimes lesions are patchy involving more than a lobule 3327

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 27:481-487, 1998-487, 1998

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

Temporal Aspects are also important in the liver

Gene Expression for Metallothionein 1a varies by time during the 24 hour day CT times; light 0 - 12: Dark 12-24

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 ?

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

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

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)

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

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

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

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

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

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

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

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

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

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 & visitors Dr. 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 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