Liver nicnas-nov-2012

Dr R B Cope 1

An Introduction to the Toxicology of the Liver & Rodent Stomach.

Rhian B. Cope BVSc BSc(Hon 1) PhD DABT ERT

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Yes, there is a lot of basic science.

It is included deliberately: if you do not understand the fundamentals of how and why the liver reacts to xenobiotics, you cannot really understand the significance and human-relevance of the changes that occur.

Understanding the mode of action is the key to just about everything in toxicology and toxicological risk assessment.

Please bear with me.

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Sections.Section 1: A Revision of the Basic Anatomy and Physiology of the Liver, Reasons for the Susceptibility of the Liver to Toxic Injury and Classical Clinical Signs of Hepatic Disease.

Section 2: Responses of the Liver to Toxic Injury

Section 3: Interpretation of Rodent Hepatic Tumour Data: The Human-Relevance Framework

Section 4: Detection/ Measurement/Assessment of Hepatic Toxicity.

Section 5: The Two Basic Classes of Hepatic Toxicants, and Classical “Must Know” Agents Causing Hepatic Damage.

Section 6: Interpretation of Rodent Stomach Tumour Data: The Human-Relevance Framework.

Section 7: Case Studies.

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Section 1.

A Revision of the Basic Anatomy and Physiology of the Liver, Reasons for the Susceptibility of the Liver to Toxic Injury and Classical Clinical Signs of Hepatic Disease.

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Learning Tasks Section 1.1. Describe and understand the toxicologically significant features of the

hepatic circulation.

2. Describe and understand the structure and toxicologically significant features of the liver lobule.

3. Describe and understand the structure and toxicologically significant features of the liver acinus.

4. Understand the toxicological significance of Kupffer, Pit and Ito cells.

5. Describe and understand the key physiological roles of the liver and the potential effects of disrupting these functions.

6. Describe and understand the toxicologically significant features of bile formation/excretion and excretion of bilirubin.

7. Describe and understand the basis for the susceptibility of the liver as a toxic target organ.

8. Describe and understand the classical clinical signs of hepatic disease.

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Hepatic Circulation and Blood Supply.

• Key points:

Liver receives blood via two routes: high oxygen blood from the hepatic artery (30%) and low oxygen blood from the portal vein (70%).

Blood leaves the liver only by the hepatic vein.

Liver is placed between venous blood returning from the bulk of the GI and peritoneal cavity and the venous arm of the systemic circulation.

WHAT ARE THE TOXICOLOGICAL CONSEQUENCES OF THIS?

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Structure of the Liver Lobule.

Low magnification view of the a liver lobule in the pig01/05/07

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Low magnification view of the human liver lobule01/05/07

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Note the lack of an endothelial basement membrane, large endothelial pores and large endocytic vacuoles. What are the key toxicological consequences of these features?

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Structure of the Liver Acinus.

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• Acinar zone 1 approximates “Periportal” using the “Lobular” system.

• Acinar zone 3 approximates “Centrilobular” using the “Lobular” system.

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Dr R B Cope 18Describe the distribution of damage (necrosis) in this liver

section using the “lobular” and “acinar” system.01/05/07


section using the “lobular” and “acinar” system.

?

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section using the “lobular” and “acinar” system.

Central Vein

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Dr R B Cope 21Centrilobular or Zone 3 Necrosis.

Central Vein

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• Hepatocytes are generated in zone 1 from their primordial stem cell and migrate from zone 1 to zone 3 before undergoing senescence/apoptosis in zone 3.

– The youngest hepatocytes occur in zone 1, the oldest occur in zone 3.

– The hepatocyte cycle in the rat is approximately 200 days.

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• All hepatocytes are NOT equal. Important functional/physiological differences occur between hepatocytes in different acinar zones.

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Hepatocyte Zonal Specialization.

LowIntermediateHighGlycogen and other nutrient stores

LowIntermediateHighNumber of mitochondria

LowIntermediateHighConcentration of materials (bile salts, bilirubin,

excreted compounds) in adjacent bile canaliculus

LowIntermediateHighLevel of fatty acid oxidation, gluconeogeneis,

and ureagenesis

HighIntermediateLowerCYP level (particularly Cyp 2E1)

Phase I predominates over Phase II

IntermediateRelatively balanced

Overall balance between Phase I and Phase II

metabolism

LowIntermediateHighBile acid excretion

LowIntermediateHighGlutathione levels

Last site of exposureIntermediateFirst site of exposure

Exposure to portal blood

LowIntermediateHighOxygen tension and level of nutrients in blood supply

Zone 3Zone 2Zone 1Parameter

Toxicological Consequences of Hepatocyte Zonal Specialization.

Glycogen and other nutrient stores

Number of mitochondria

Concentration of materials (bile salts, bilirubin,

excreted compounds) in adjacent bile canaliculus

Level of fatty acid oxidation, gluconeogeneis,

and ureagenesis

CYP level (particularly Cyp 2E1)

Overall balance between Phase I and Phase II

metabolism

Bile acid excretion

Glutathione levels

Exposure to portal blood

Oxygen tension and level of nutrients in blood supply

Zone 3Zone 2Zone 1Parameter

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Kupffer Cells.

• Kupffer cells are the resident tissue macrophage of the liver. Located in the sinusoids.

• Large number of Kupffer cells are present in the liver: 80% of body’s resident tissue macrophages.

• Fully functional macrophage: can trigger inflammation and act as antigen presenting cells.

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Kupffer Cells.

• Of considerable importance in hepatic toxicology:

– Activation during inflammation results in the generation of various free radicals e.g. superoxide anion, peroxynitrite, nitrogen oxides

– Triggering and participation in inflammation.

– Accumulation of iron (hemosiderin, ferritin).

– Degradation of heme.

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Pigment accumulation within Kupffer cells.01/05/07

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Pit Cells.

• Located in the space of Disse.

• Function as NK or LAK cells.

• Important in inflammation.

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Ito Cells.

• Synonyms = “fat cells”, stellate cells.

– Two major roles:

• Storage of Vitamin A.

• During inflammation or liver damage, produce collagen i.e. responsible for hepatic fibrosis.

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Congestive cirrhosis (replacement of hepatocytes with fibrous tissue) secondary to right sided heart failure, trichrome stain. Remember: Ito cells are responsible for the laying down of new collagen within the liver. WHAT ARE THE CRITICAL FUNCTIONAL CONSEQUENCES OF SUCH A REACTION IN THE LIVER?

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A Concise Summary of Key Hepatic Functions

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Consequences of Disruption of Hepatic FunctionConsequences

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Bile Formation and Hepatic Excretion.• Bile formation involves both hepatocytes and

cholangiocytes

• Bile formation involves 8 basic processes:

1. Materials that undergo biliary excretion move from the sinusoid through the space of Disse and through the basolateral hepatocyte cell membrane via diffusion, active transport or endocytosis.

2. The materials for excretion are transported across the hepatocyte with or without metabolism and storage and then actively transported into the canaliculi.

3. Vesiclular transport involves the detachment of lipid vesicles from the apical hepatocyte membrane to form bile micelles. Bile micelles contain lipophilic compounds, bile salts, cholesterol, phospholipids, and high molecular compounds

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Bile Formation and Hepatic Excretion.4. Excretion of compounds is sufficient to generate osmotic water

flow into the bile canaliculi.

5. Forward movement of bile within the canaliculi occurs by ATP-dependent peristaltic contraction of the actin-myosin web located underneath the apical membrane of the hepatocytes.

6. Within the bile ductules and common hepatic duct, bile composition and volume are modified by cholangiocytes:

7. Volume increases due to the osmotic gradient created by the active excretion of HCO

3- in exchange for Cl- by cholangiocytes;

~ 40% of bile volume is due to this excretion mechanism.

8. Cholangiocyte re-uptake of some constituents (some bile acids) occurs.

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Bile Formation and Hepatic Excretion.

• Molecules with a molecular weight of ≤ 300 Da are more efficiently excreted in bile than molecules with a greater molecular weight.

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Major Hepatocyte and Cholangiocyte Transporters involved in Bile Formation

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Major Hepatocyte Involved in Bile Formation

Bilirubin, sulfobromophthalein; inhibited by phenylmethyl-sulphonyl fluoride; exists in two metastable forms: high and low affinity.

Bilitranslocase

Divalent lipophilic cations, xenobiotics that contain a tertiary or quarternary amine group

Organic cation transporter I (OCT I)

Uptake of amphiphilic compounds, steroid conjugates, neutral steroids, sulfobromophthalein (OATP2), bilirubin (OATP2), glutathione conjugates, leukotriene s, C4

organic cations, small peptides, digoxin

Organic anion transporter polypeptide (OATP)

Uptake of conjugated bile acids, estrogens

Na+-taurocholate-co-transporting peptide (NTCP)

FunctionBasolateral Transporters

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Major Hepatocyte Transporters Involved in Bile Formation.

Uptake of indocyanine green, and nonsteroidal anti-inflammatory drugs, such as ketoprofen, indomethacin, and salicylates through the basolateral hepatocyte cell membrane

Organic anion transporter 2 (OAT2)

FunctionBasolateralTransporters

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Major Hepatocyte Transporters Involved in Bile Formation.

Excretion of glucuronic acid, sulfate and glutathione (anionic) conjugates, phospholipids; Excretion of mono- and diglucuronic acid bilirubin conjugates (MRP2) and glutathione-sulfobromophthalein conjugates (MRP2)

Multidrug resistance-associated proteins (MRP); MRP2 = canalicular multispecific organic anion transporter (cMOAT)

Transports monoanionic bile salts.SPGP = bile salt export pump (BSEP)

Excretion of cationic and lipophilic compounds. MDR1 has no physiological substrate in non-ruminants; function is the secretion of amphiphilic cationic xenobiotics, steroid hormones, hydrophobic pesticides and glycolipids; responsible for phyloerythrin excretion in ruminants!

Multidrug resistance proteins (MDR), particularly MDR1(Note: MDR1 = p-glycoprotein, which has now been renamed the ATP-binding cassette sub-family B member 1 transporter, or ABCB1)

FunctionApical Transporters

Hepatic Bilirubin Excretion.Heme containing proteins (Hb, Mb, CYP450)

Free heme (red)

Biliverdin (green)

Heme oxygenase

Spleen, K

upffer cells,

Reticuloendothelial system

Bilirubin (Br;brown)

Biliverdin reductase

Albumen (ALB)

Alb-Br

(“Free” or unconjugated Br)

Systemic Circulation

Alb-Br

Alb

Br

BT*Br

Sin

usoi

d

Spa

ce o

f D

isse

Bile

can

alic

ulus

*Organic anion transport protein; *Bilitranslocase; * Rate limiting step for bilirubin excretion

Hepatocyte

Conjugated Br

(Gluc-Br)

UD

P-glucuronide

MRP2*

Gluc-Br in Bile

UG

T-1A1

OATP*

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Extrahepatic Aspects of Bilirubin Excretion.

• Conjugated bilirubin excreted in the bile is converted by bacterial action within the ileum and colon into urobilinogen which undergoes enterohepatic circulation.

• Urobilinogen that is not taken up and re-excreted by the liver passes into the systemic circulation and is excreted by the glomerular filtration in the kidneys

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Extrahepatic Aspects of Bilirubin Excretion.

• The amount of urobilinogen formed, and thus excreted by the kidneys increases dramatically with increased formation of bilirubin (e.g. hemolysis).

• The amount of urobilinogen in urine will decrease with:– Severe cholestasis (failure of conjugated bilirubin

excretion).– Bile duct obstruction.– Severe disruption of the GI microflora (antibiotics).

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Important Aspects of Bilirubin Excretion.

• The excretion of conjugated bilirubin is inhibited by the administration of sulfobromophthalein due to competition for the MRP2 transporter.

• Impaired hepatic sulfobromophthalein excretion (i.e. increased or delayed retention) has at least three potential causes:

– Cholestasis due to impaired apical excretion.– Inhibition of glutathione-S-transferases (requires

conjugation to glutathione for excretion).– Impaired basloateral bilitranslocase and OATP function.

* note: bromosulfonphthalein (BSP) was a commercial brand name for sulfobromophthalein. Older literature will often refer to a BSP test which simply means a test for plasma clearance of sulfobromophthalein.

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• Bilirubin in plasma is measured by the van den Bergh assay which makes two different measurements: total bilirubin and direct bilirubin.

• Classically, the direct bilirubin is regarded as a measure of conjugated bilirubin in plasma.

• Indirect bilirubin (unconjugated) = total bilirubin – direct bilirubin.

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• Modern analytical methods have now demonstrated that plasma from normal individuals contains virtually no conjugated (i.e. “direct”) bilirubin.

• Elevations of plasma direct or conjugated bilirubin primarily occur with:– Obstruction of the bile ducts or canaliculi.– Decreased canalicular contraction.– Inhibition of MRP2.– Hepatocellular disease.

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Bilirubin Excretion in the Neonate.

• Bilirubin excretion, like most hepatic excretion, takes time to develop in neonates.

• Bilirubin produced by the fetus is cleared by the placenta and eliminated by the maternal liver.

• After birth, the neonatal liver slowly develops the capacity for bilirubin clearance and excretion.

• Levels of UGT1A1 in neonatal hepatocytes are low and unconjugated bilirubin is excreted into the gut.

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• The neonatal gut lacks the microflora to convert bilirubin to urobilinogen and bilirubin undergoes enterohepatic cycling.

• Levels of MRP2 are also low in the neonate. Remember transport of conjugated bilirubin across the hepatocyte apical cell membrane is the rate-limiting step for bilirubin excretion.

• Neonates typically have elevated free bilirubin in their plasma due to impaired excretion by MRP2 and enterohepatic cycling.

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• Any xenobiotic that increases the production of bilirubin in the neonate will produce rapid, large increases in plasma bilirubin. – Any agent that produces hemolysis or defective

erythrogenesis.– Any agent that produces hemorrhage.– Any agent that produces cholestasis.

• This results in a condition called kernicterus (bilirubin encephalopathy) in which bilirubin crosses the blood-brain barrier and precipitates within the basal ganglia and other sites in the brain resulting in CNS damage. Yellow staining of brain nuclei due to bilirubin precipitates is the classical pathology associated with kernicterus.

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Globus pallidus staining with bilirubin

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Basis for the Susceptibility of the Liver to Toxicity.

• Position within the circulatory system: high exposure to xenobiotics absorbed via the GI (also peritoneum) i.e. first pass effect.

• High level of biotransformation, and therefore, significant risk of generating reactive metabolites.

• Susceptibility to oxidant injury.

• Susceptibility to hypoxic injury (centrilobular).

• Critical biosynthetic/homeostatic functions.

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• Ability to concentrate xenobiotics within the biliary tree,

• Large tissue macrophage population: inflammation and oxidative injury.

• Little or no selectivity of sinusoidal endothelium (large pores).

• Capacity to separate xenbiotics from albumen and other carrier proteins.

• Capacity to accumulate metals, vitamin A and other xenobiotics.

• Liver has high energy consumption and Is susceptible to agents that affect mitochondrial function.

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• Enterohepatic circulation can result in sustained exposure to xenobiotics.

• Lipophilic xenobiotics tend to concentrate within the liver since it is relatively rich in cell membranes

• Substrates for the transporter systems of the basolateral hepatocyte membrane also tend to selectively accumulate in the liver e.g. phalloidin, microcystin.

• Compounds that have hepatic storage can cause toxicity e.g. iron (stored as ferritin), cadmium (stored as a Cd-metallothionine complex), vitamin A (selectively stored in Ito cells)

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Patients showing clear evidence of jaundice: yellow discoloration of the skin and sclera.

Important differential is high dietary beta carotene – tissues and skin are stained yellow, but the sclera remains white!

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Clinical Signs of Acute Hepatocellular Disease.

• Markers of malaise i.e. fatigue, weakness, nausea, poor appetite.

• Icterus/jaundice: probably the best clinical marker of severity. Indicates bilirubin level > 2.5 mg/dl.

• Spider angiomata and palmar erythema.

• Itching (self mutilation in animals).

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Clinical Signs of Acute Hepatocellular Disease.

• Right upper quadrant abdominal pain.

• Abdominal distention.

• Intestinal bleeding.

• ± Heatomegaly.

• Bilirubinuria: dark characteristically colored urine

• In many cases of hepatocellular disease, there are no clinical signs. Cases are recognized by biochemical liver tests.

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Clinical Signs of Advanced or Chronic Hepatocellular Disease.

• Weight loss, muscle wasting.

• Evidence of hemorrhage and coagulopathy.

• Ascites.

• Edema of the extremities.

• Fetor hepaticus = typical sweet ammoniacal odour of patients with hepatic failure (failure of ammonia clearance/metabolism).

Evidence of inadequate serum protein synthesis.

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Ascites following severe liver disease. Note the eversion of the umbilicus.

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Mid-level abdominal CT scans. Left = normal; Right = ascites secondary to liver failure.

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• Hepatic encephalopathy (change in sleep patterns, change in personality, irritability, mental dullness, disorientation, stupor, asterixis*, flapping tremors of body and tongue, coma).

• Caput medusa = development of prominent collateral veins radiating from the umbilicus due to the recanulation of the umbilical vein and its tributaries due to portal hypertension and porto-systemic shunting.

* Asterixis = a motor disturbance marked by intermittent lapse of an assumed posture due to intermittent sustained contraction of muscle groups; characteristic of hepatic coma; often assessed by asking the patient to write or draw simple pictures (e.g. draw a clock face).01/05/07

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Caput medusae associated with portal hypertension, portosystemic shunting and

severe liver disease.01/05/07

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• Hepatorenal syndrome: characterized by progressive renal failure that develops following chronic liver disease + ascites and other evidence of liver failure. Mechanism is unknown but the syndrome is associated with altered renal hemodynamics and altered prostaglandin levels are implicated.

• Portal hypertension, portosystemic shunting and acute venous hemorrhage due to rupture of abdominal veins.

• Spontaneous bacterial peritonitis (failure of bacterial opsonization due to low albumen and other opsonizers).

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• Hepatopulmonary syndrome: development of right to left intrapulmonary shunts in advanced liver disease. Mechanism is unknown but involves altered pulmonary nitric oxide levels.

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Clinical Signs of Advanced Hepatocellular or Cholestatic Disease in Ruminants: Secondary Photosensization.

In ruminants:

Chlorophyll PhylloerythrinRumen bacteria

Absorbed

Hepatocyte

Transported across the apical hepatocyte cell membrane by ATP-binding cassette transporter B1 [p-glycoprotein or MDR 1)

Excreted in bile

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Clinical Signs of Advanced Hepatocellular or Cholestatic Disease in Ruminants: Secondary Photosensitization.

• Prolonged inhibition of ABCB1, cholestasis or hepatocelular disease in ruminants results in an accumulation of phylloerythrin within the circulation and tissues.

• Phylloerythrin absorbs light and acts as a photosensitizer within the skin resulting in severe skin inflammation and sloughing.

• Disease in sheep (particularly associated with sporodesmin-induced liver disease) is colloquially called “facial eczema.”

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Secondary photosensitization of the face due to sporodesmin poisoning in a sheep01/05/07

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Severe secondary photosensitzation of the udder of a cow with advanced hepatic disease (again due to sporodesmin)

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Section 2:

Responses of the Liver to Toxic Injury.

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Learning Tasks Section 2.

1. Describe and understand the stereotypical cellular responses of the liver to xenobiotic injury.

2. Describe and understand the processes involved in the development of cholestasis.

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Stereotypical Responses of the Liver to Toxicant Injury.

• The patterns of the hepatocellular response to toxicant injury are generally stereotypical and not toxicant specific (although there are exceptions to this rule).

• The hallmark of the liver’s response to toxicant injury is its large functional reserve and large capacity for healing, often with no significant sequelae!– For example, a 2/3 hepatectomy is survivable and both

normal liver function and size will be restored within weeks!

– This will occur provided significant fibrosis or massive necrosis of the lobules does not occur and the source of injury is removed i.e. exposure is not chronic.

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Hepatocellular Adaptive Responses.

• These changes are generally reversible once xenobiotic exposure stops.

• In terms of a toxicology study, ideally this propensity for reversal should be tested by the inclusion of an adequate post-exposure recovery period in the study.

• This inevitably involves inclusion of additional experimental groups i.e. groups that is euthanitized at the end of exposure (necropsy + histology) plus groups that are euthanitized 14 to 30 days post exposure + appropriate control groups.

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• Sadly this is rarely done despite the provision for this in the OECD guidelines.

• My personal view is that histological discrimination of the types of lesion present is not sufficient to claim reversibility; must have actual documented study evidence of the reversibility of hepatic adaptive responses!

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• Represent adaptive responses to xenobiotic response rather than hepatocellular damage per se.

• Used as histological markers of xenobiotic exposure.

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• Do not result in disease per se but are often of significance for the toxicokinetics/toxicodynamics of drugs and other xenobiotics and thus may significantly influence the toxicity of particular toxins/toxicants.

• Usually detected histologically but may be visible grossly as hepatomegaly and/or increased liver weight.

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Hepatocellular Adaptive Responses: Centrilobular Hepatocellular Hypertrophy.

• Due to ↑ smooth endoplasmic reticulum content in centrilobular/Zone 3 hepatocytes.

• Associated with chemical induction of CYP, particularly CYP2E1.

• Associated with massive increases in the amount of smooth endoplasmic reticulum.

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Hepatocellular Adaptive Responses: Centrilobular Hepatocellular Hypertrophy.

• Reversible following removal of the initiating agent.

• Example initiating agents: phenobarbital and other oxybarbiturates, Ah receptor agonists (TCDD, PCDFs).

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Centrilobular hepatocyte hypertrophy in a mouse treated with phenobarbital for 8 months.

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Centrilobular hepatocyte hypertrophy in a mouse treated with phenobarbital for 8 months Note the eosinophilic

cytoplasm due to the large increase in smooth endoplasmic reticulum as a result of CYP (particularly CYP2E1) induction.01/05/07

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Hepatocellular Adaptive Responses: Eosinophilic Centrilobular Hepatocellular Hypertrophy.

• Due to ↑ peroxisomes in centrilobular hepatocytes.

• Prolonged eosinophilic centrilobular hypertrophy is associated with pericanalicular lipofuscin pigment deposition.

• Prolonged exposure to chemicals that induce peroxisome induction may result in hepatocellular neoplasia in rodents.

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Hepatocellular Adaptive Responses: Eosinophilic Centrilobular Hepatocellular Hypertrophy.

• Reversible following removal of the initiating agent.

• Classical agents: phthalate plasticizers.

• Rodent-specific response.

• Relevance to humans is controversial!

• Currently regarded as not relevant to humans in many jurisdictions, however this is an area of considerable scientific challenge

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Centrilobular eosinophilic hepatocyte hypertrophy (left) in a mouse due to chronic exposure to phthalates. Right image shows immunohistochemical staining for peroxisomes. Note that chronic exposure to peroxisome proliferators is carcinogenic in rodents but not humans.01/05/07

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Hepatocellular Adaptive Responses: Xenobiotic-Induced Hepatocyte Hyperplasia.

• Usually accompanied by CYP induction, hepatomegaly, and hepatocyte hypertrophy.

• Never continues for more than a few days.

• Reversible following removal of the initiating agent. Reversion is associated with ↑ hepatocyte apoptosis.

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Derived from the UK PSD guideline (included as an appendix to the notes)

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Early Markers of Hepatocellular Damage: Hepatocyte Nucleolar Lesions.

• Due to changes in RNA synthesis.

• Changes include: ↓ size, ↑ size, nucleolar fragmentation, nucleolar segregation.

• ↓ Nucleolar size is usually an acute lesion that occurs within hours of hepatotoxin exposure; often the first identifiable toxic hepatic lesion.

• ↑ Nucleolar size is commonly associated with hepatic neoplasia.

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Early Markers of Hepatocellular Damage: Hepatocyte Polysome Breakdown.

• In normal protein synthesis, ribosomes are evenly spaced along single strands of mRNA forming a structure called a polysome.

• ↓ RNA synthesis ↓ polysomes loss of basophilic granules in hepatocyte cytoplasm.

• Loss of basophilic granules in hepatocyte cytoplasm implies ↓ cellular protein synthesis and is an early marker of hepatocellular injury.

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Reversible Hepatocellular Injury: Hydropic Degeneration.

• Accumulation of water in the cytosol or rough endoplasmic reticulum.

• Characterized histologically by pale-staining cytoplasm, narrowing of the sinusoids and space of Dissė.

• Typically reversible.

• Due to failure of hepatocytes to maintain intracellular Na+ balance.

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Hepatocyte hydropic degeneration.

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Reversible Hepatocellular Injury: Hepatic Lipidosis (“Fatty Liver”).

• Two basic forms: Accumulation of triglycerides or accumulation of phospholipids.

• Responses are non-specific: many other conditions cause fatty liver and it is NOT pathognomonic for hepatotoxicity.

• Accumulation of triglycerides within membrane-bound vesicles in hepatocytes

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• Occurs due to an imbalance in the uptake of fatty acids and their excretion as very low density lipoproteins (VLDL) due either to impaired VLDL synthesis or secretion.

• Typically associated with acute exposure to many hepatotoxins.

• Typically reversible and usually does not involve hepatocellular death.

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Fatty liver due to triglyceride accumulation.

– Triglycerides are located within membrane-bound cytoplasmic vesicles.

– Occurs due to an imbalance in the uptake of fatty acids and their excretion as very low density lipoproteins (VLDL) due either to impaired VLDL synthesis or secretion.

– Typically associated with acute exposure to many hepatotoxins.

– Typically reversible and usually does not involve hepatocellular death.

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Fatty liver due to phospholipid accumulation.

– Caused by toxins that bind to phosopholipids and block their catabolism.

– Phosopholipids accumulate in hepatocytes, Kupffer cells and extrahepatic cells.

– Affected cells have foamy cytoplasm.

– Lesion is reversible and does not involve cell death.

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Human liver. Fatty change due to alcohol. Note the color. Surface will feel “greasy”.

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Hepatocyte fatty change due to ethanol exposure. Note: fat droplets appear clear due to their extraction

during tissue processing.01/05/07

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Fine needle aspirates of hepatocytes. Normal on the left, fatty change on the right.01/05/07

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Hepatocellular Death: Hepatocellular Apoptosis and/or Necrosis.

• Both apoptosis and necrosis occur and these endpoints can often be regarded as points on a dose response curve i.e. apoptosis for low exposures, necrosis for high exposures.

• Toxins are generally specific for a single area or zone within the hepatic lobule, although this pattern can be altered by dose and duration of exposure.

• The significance of necrosis as an endpoint in the liver is that it almost always occurs with inflammation which tends to amplify the amount of damage that occurs.

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Hepatocellular Death: Centrilobular, Zone 3 or Periacinar Necrosis.

• Most common reaction to toxic injury.

• Lesion is usually uniformly distributed within the liver.

• Typically, cellular injury is typically limited to hepatocytes but destruction of the endothelium and centrilobular hemorrhage may also occur.

• Generally rapidly repaired with minimal fibrosis in the area surrounding the central vein.

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• Centrilobular necrosis can be triggered by ↓ blood flow since this is the area of the lobule that receives blood last, is the most hypoxic and is the most nutrient-limited.

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• Metabolic basis for the pattern (i.e. metabolic zonation) is that the centrilobular hepatocytes have the highest levels of CYP and therefore the highest activation of xenobiotics to potentially toxic metabolites.

• – In this area, phase I and phase II metabolism are out of

balance.

– Phase I metabolism often converts xenobiotics to electrophilic metabolites. Phase II metabolites are usually stable and non-reactive.

– If phase I predominates over phase II metabolism, the tendency for production/accumulation of reactive electrophilic metabolites is higher, thus there is a greater tendency for hepatocellular injury.01/05/07

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Centrilobular necrosis.

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Hepatic centrilobular necrosis.01/05/07

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Hepatocellular Death: Periportal or Zone 1 Necrosis.

• Less common than centrilobular necrosis.

• Hemorrhage is rarely associated with periportal necrosis.

• Inflammatory response is usually very limited or absent.

• Repair is usually rapid with minimal fibrosis.

• Repair is often accompanied by bile ductule proliferation which usually regresses over time.

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Hepatocellular Death: Periportal or Zone 1 Necrosis.

• Pathophysiological basis for periportal necrosis.

• Periportal area receives blood first and is thus the first area to be exposed to xenobiotics and is also exposed to the highest concentration of xenobiotics.

• Metabolic zonation effects: area has the highest oxygen tension.

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Periportal degeneration and portal cirrhosis.01/05/07

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Hepatocellular Death: Massive or Panacinar Necrosis.

• Massive wide-spread death of hepatocytes with only a few or no survivors.

• Involves the whole lobule; not all lobules are equally affected.

• Necrosis extends from the central vein to the portal area (bridging necrosis).

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Hepatocellular Death: Massive or Panacinar Necrosis.

• Severe panacinar necrosis and destruction of the supporting structures usually results in ineffective repair i.e. variably sized regenerative nodules that lack normal lobar structure; significant permanent fibrosis usually occurs.

• Usually occurs following exposure to massive doses of hepatotoxins or when toxins are directly injected into the portal venous system.

• In the case of intravascular injection of the toxin, massive necrosis may be confined to specific liver lobes due to incomplete mixing of the agent in the portal vascular supply.

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Hepatic massive necrosis. Note the periportal accumulation of bile pigments.

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Hepatic massive necrosis.01/05/07

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Cirrhosis.

• Cirrhosis = hepatic fibrosis + nodular regeneration.

• 2 basic forms:– Centrilobular (i.e. inside outside fibrosis). Usually

occurs secondary to chronic right sided heart failure and/or hepatic vein hypertension.

– Periportal (i.e. outside inside fibrosis). Usually occurs secondary to repeated episodes of hepatocellular necrosis or following an episode of massive necrosis or chronic/significant damage to the sinusoidal vasculature.

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Nodular regeneration and periportal cirrhosis following massive necrosis. Trichrome stain. Note that the regenerating liver nodules vary in size and are highly

disorganized. There is no regular lobular structure and extensive periportal fibrosis is present. What do you think the functional consequences this lesion are?

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Cirrhosis.

• Regenerating hepatocyte lobules nodules do not have the normal lobular structure and vary in size. Inevitably hepatic function is significantly compromised.

• Irreversible, usually progressive and typically has a poor prognosis.

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Hepatocyte Megalocytosis.

• Characterized by the appearance of large multinucleate hepatocytes in areas of hepatocellular regeneration.

• Megalocytes are hepatocytes that have undergone cell division but cannot complete cell separation.

• Sign of frustrated or ineffective hepatocyte proliferation i.e. suggests a blockage in the cell division process.

• Classically associated with the pyrrolizidine alkaloids, but also occur with several hepatic carcinogens.

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Bile Duct Hyperplasia.

• Common response to xenobiotics.

• May be restricted to the periportal area or may extend beyond the periportal area.

• Simple bile duct hyperplasia is not associated with cholangiofibrosis.

– May remain static, regress or progress.

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Bile Duct Hyperplasia.

• Cholangiofibrosis.– Characterized by proliferation of bile ducts

surrounded by fibrous tissue.

– May regress over time following removal of the initiating agent but is generally regarded as a more serious type of injury due to the fibrosis.

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Periportal Bile duct hyperplasia.

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Hepatocellular Death: Focal Necrosis.

• Randomly distributed death of single or small clusters of hepatocytes.

• Uncommon.

• Usually accompanied by mononuclear cell infiltration at the lesion site.

• Pathophysiological basis for the lesion is poorly understood.

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Damage to the Sinusoidal Epithelium: Peliosis Hepatis and Related Syndromes.

• Progressive damage to the sinusoidal endothelium results in eythrocyte adhesion, eventual blockage of the sinusoidal lumen and hepatic engorgement.

• Typically associated with pyrrolizidine alkaloids.

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Damage to the Sinusoidal Epithelium: Peliosis Hepatis and Related Syndromes.

• Peliosis hepatis: characterized by clusters of greatly dilated sinusoids that occur randomly through the liver parenchyma.

• Occasionally associated with other toxins that damage the hepatic endothelium, but also occurs spontaneously in rodents

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Lesions of Ito Cells: Ito Cell Hyperplasia and Spongiosis Hepatis .

• Enlargement is associated with hypervitaminosis A.

• Ito cell proliferation is often associated with centrilobular injury; under these circumstances, Ito cells produce collagen and are responsible for inside outside cirrhosis.

• Spongiosis hepatis.– Found only in rodents.– Due to proliferation of abnormal Ito cells.– Due to aging or exposure to hepatocarcinogens.

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Lesions of Kupffer Cells: Iron, Endotoxin and Ricin.

• Kupffer are the primary site of iron storage in the liver and damage occurs with iron overload.

• Kupffer cells are the primary site of uptake of endotoxin/LPS in the liver. This may result in Kupffer cell activation and secondary damage to hepatocytes due to inflammation or death of the Kupffer cells.

• Kupffer cells are preferentially damaged by ricin.

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Hepatocellular Pigmentation.

• Glycogen accumulation.– Appears as a clear cytoplasm with indistinct

vacuoles; identifiable using periodic acid-Schiff (PAS) staining.

– Due to either up-regulation of glycogen synthesis or impaired glycolysis.

• Lipofuscin.– Normally accumulates with aging, but ↑ deposition

occurs following exposure to peroxisome proliferators.

– Stains brown with H & E; special stain is Schmorl's stain; autofluoresces under UV light.

– Lipofuscin is due to the lysosomal accumulation of partially digested lipids.

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• Ferritin/hemosiderin.

– Excess iron is stored as ferritin (conjugate of iron + apoferritin) or hemosiderin (incomplete breakdown product of ferritin) in membrane bound granules (siderosomes) particularly in Kupffer cells.

– Appears as golden brown granules in H & E sections; special stain is Pearl’s Prussian blue.

– Often has a pericanalicular distribution.

– Due to excessive iron intake, excessive erythrocyte destruction or some hepatotoxins.

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• Copper.

– Appears as enlarged hyperchromatic hepatocytes + necrosis + granulocytic/monocytic infiltrate.

– Special stains are rubeanic acid or rhodamine.

– May also be associated with Mallory body formation (Mallory bodies are red globular accumulations in the cytoplasm which are composed of cytoskeletal filaments).

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Oval Cell Hyperplasia.

• Response is peculiar to rodents; Extensive oval cell hyperplasia is only rarely observed in non-rodent species.

• Oval cells are presumed to be hepatocyte stem cells.

• Occurs under two circumstances:

– Hepatocyte proliferation following hepatocyte necrosis.• Oval cells are most numerous when hepatocyte

regeneration is partially or completely blocked e.g. with repeated insults or chronic exposure to a toxicant.

– Exposure to hepatic carcinogens.

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Oval Cell Hyperplasia.

• Can occur independently or concurrently with bile duct hyperplasia.

• Response is always regarded as potentially neoplastic.

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Oval cell hyperplasia in a mouse exposed to a hepatic carcinogen.

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Hepatic Neoplasia.

• Involves hepatocellular neoplasia, bile duct neoplasia, endothelial neoplasms and Kupffer cell neoplasms.

• Very common reaction to many carcinogens in rodent toxicology models:

– ~ 50% of carcinogens cause hepatic neoplasia in rodents.

– This is significantly different from humans where hepatic neoplasia is relatively uncommon: this remains a significant area of controversy and concern in terms of risk analysis and regulatory toxicology. Are agents that produce rodent liver tumors really of great significance to humans?? (Answer: depends on the mechanism)

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Hepatic Neoplasia.• Hepatocyte neoplasias.

• Marked strain difference in rate of spontaneous hepatocellular carcinomas in rodents (~ 30 – 50% incidence in C3H mice versus < 5% in male C57B1/6 mice)

• Malignant hepatocyte neoplasias = hepatocellular carcinomas.

• Benign hepatocyte neoplasias = hepatocellular adenoma.

• Nodular hyperplasia = benign hepatocyte proliferative lesion which is reversible once the initiating agent is removed in some (but not all) cases.

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Hepatic Neoplasia.

• Bile duct neoplasia.• 3 types: cholangiocarcinoma (malignant),

cholangiofibroma (benign), cholangioma (benign).

• The 3 different types represent a single continuous spectrum of lesions.

• Chemicals that induce bile duct hyperplasia usually fail to cause bile duct neoplasia i.e. bile duct hyperplasia is NOT a preneoplastic condition.

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• Cholestasis

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Classification of Cholestasis.

• Definable at 3 levels: biochemical, physiological and morphological.

• Biochemical cholestasis.– Hallmark is ↑ level of bile constituents in serum i.e. ↑

conjugated bilirubin, ↑ serum bile acids.

• Physiological cholestasis.– ↓ bile flow due to decrease in canalicular

contraction.

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• Morphological cholestasis.

– Hallmark is the accumulation of bile pigment in canaliculi or hepatocytes, often accompanied by deformation and/or loss of canalicular microvilli.

– Typically has a centrilobular distribution.

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Morphological cholestasis in mice chronically treated with phenobarbital. Note the predominantly intracellular accumulation of bile pigments. What basic mechanism does this pathology suggest? What other changes are present? What is the distribution of this lesion?01/05/07

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Gross morphology of human liver showing evidence of cholestasis: note the color.

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• An alternative system of classification is based on the presence or absence of evidence of damage to bile ducts:

• Canalicular cholestasis: not associated with destruction of cholangiocytes and therefore, serum alkaline phosphastase (ALP) levels are normal.

• Cholangiodestructive cholestasis/Acute bile duct necrosis.– Associated with ↑ serum ALP.– Associated with destruction of cholangiocytes,

portal inflammation, bile duct proliferation and portal fibrosis.

– Usually associated with rapid replacement of the bile duct epithelium.

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Mechanisms of Cholestasis.

• There are at least 6 potential mechanisms of cholestasis:

– Impaired uptake of bile precursors through the hepatocyte basolateral cell membrane. e.g. estrogens ↓ the Na+/K+ ATPase necessary for bile salt transport across the hepatocyte basolateral cell membrane.

– ↓ transcytosis of bile precursors through the hepatocyte cytoplasm. e.g. microcystin disrupts the hepatocyte cytoskeleton which ↓ transcytoplasmic vesicular transport and hepatocyte deformation.

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Mechanisms of Cholestasis.

– Impaired hepatocyte apical secretion. e.g. estrogens inhibit transport of glutathione conjugates and bile salts.

– ↓ Canaliculus contractility.

– ↓ Integrity of bile canalicular tight junctions.

– Concentration of reactive species in the bile canaliculus and resultant damage to cholangiocytes and/or hepatocytes. This mechanism is probably the most common.

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Section 3:

Rodent Liver Tumours and Human Health Risk Assessment

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1. Understand and recognize the types of pre-neoplastic lesions present in the rodent liver and their implications in terms of carcinogenesis and risk assessment.

2. Understand the fundamental differences between adenomas and carcinomas.

3. Understand the mode of action of human hepatic carcinoma.

4. Under the ILSI/HESI mode of action framework for interpretation of rodent liver tumour data for human risk assessment.

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(A)Low-power photomicrograph of an focus of hepatocellular alteration (FHA) in a control mouse, which is recognizable as dysplastic under higher power (magnification, 63; bar = 100 µm).

(B)Higher magnification of FHA in (A) illustrating dysplasia including nuclear enlargement, increased nuclear/cytoplasmic ratio, nuclear hyperchromasia, variation in nuclear size and shape, irregular nuclear borders, and nucleoli that are increased in size and number with irregular borders (magnification, 250; bar = 100 µm).

Progression to Neoplasia: Dichloroacetic Acid (DCA)

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(C) Large FHA in a liver from a mouse treated with 1 g/L DCA; note irregular border and lack of compression at edge (magnification, 63; bar = 100 µm).

(D) Higher magnification of FHA in (C) illustrating a focus of dysplastic cells within the LFCA (magnification, 400; bar = 100 µm).


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(E) Edge of a large area of dysplasia (AD) from a mouse treated with 3.5 g/L DCA, demonstrating compression of adjacent parenchyma and "pushing" border of lesion (magnification, 63; bar = 100 µm).

(F) Higher magnification of AD in (E) illustrating dysplastic cells (magnification, 400; bar = 100 µm).


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Carcinoma


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Foci of Hepatocellular Alteration: “Pre-neoplastic” change

• Society of Toxicologic Pathology Classifications:

• Foci of hepatocellular alteration:• Basophilic cell foci, tigroid type and homogenous type –

increased RER and decreased cell glycogen;

• Eosinophilic (acidophilic) cell foci – deficient in glucose-6-phosphatase; ground glass appearance;

• Clear cell foci – large unstained cytoplasm with no vacuoles;

• Amphiphilic cell foci – intensely eosinophilic cytoplasm;

• Mixed cell foci.

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Basophilic FHA

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Eosinophilic FHA

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Clear Cell FHA

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Mixed FHA

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• Occur spontaneously with age in rats; also occasionally in dogs & non-human primates;

• Type and number of spontaneous foci vary with strain;

• Have the characteristics of initiated ± promoted cells;

• Number increase with exposure to genotoxic carcinogens;

• Represent an “adaptation” of the hepatocytes to a hostile environment i.e. maladaptive response;

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• Often express placental glutathione S-transferase (GST-P) and are UDP-glucuronosyltransferase negative in rats. Variable expression patterns found in mouse foci;

• Elevated replicative DNA synthesis;

• Altered expression of various growth factors;

• Over responsive to mitogens;

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• Over responsive to mitogens

• Inherent defects in growth control (i.e. becoming autonomous in terms of growth)

• Genomic instability

• Aberrant methylation of p16 TSG

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• Mutations of ß-catenin

• Decreased apoptosis;

• Clonal origin demonstrable in vitro

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GST-P Positive FHA

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• Relevance to humans:

• Similar pre-neoplastic foci occur in humans exposed to hepatic carcinogens (both viral and chemical);

• Also occur with non-genotoxic hepatocarcinogens i.e. anabolic steroids;

• Potentially relevant to humans depending on the mechanism/mode of action!

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• Reversibility:

• In the case of chemically stimulated FHA’s, a high proportion will partially or near-completely regress when the stimulus is removed;

• Meet the criteria for “initiation + promotion”;

• Initiation is irreversible, but initiation is not phenotypically detectable;

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FHA Versus Focal Nodular Regenerative Hyperplasia and Nodular Regenerative Hyperplasia

• Key differences:

• Cells phenotypically normal;

• Circumscribed i.e. not invading surrounding normal tissue;

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FHA Versus Focal Nodular Regenerative Hyperplasia and Nodular Regenerative Hyperplasia

• Key differences:

• May be divided into pseudolobules by fibrous tissue (focal nodular regenerative hyperplasia);

• Not pre-neoplastic.

– BUT: Can be very difficult to distinguish from FHA!

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Foci of Pancreatic Tissue

• Metaplasia NOT neoplasia;

• Islands of seemingly “normal” exocrine pancreatic tissue within the liver;

• Induced by Arochlor1254 i.e. Ah-receptor mediated phemnomenon;

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Focal hepatocyte adenoma

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Adenoma Acinar Type(An adenoma is a benign tumor (-oma) of glandular origin)

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Adenoma Trabecular Type(An adenoma is a benign tumor (-oma) of glandular origin)

01/05/07

http://www.ncbi.nlm.nih.gov/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=An%20external%20file%20that%20holds%20a%20picture,%20illustration,%20etc.,%20usually%20as%20some%20form%20of%20binary%20object.%20The%20name%20of%20referred%20object%20is%20ch9f2.jpg.%20&p=BOOKS&id=9548_ch9f2.jpg

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Adenoma – Human Vs Rodent• Rodent

• Clearly distinguishable from regenerative hyperplasia;

• Usually larger than one lobule;

• Compress the surrounding tissue;

• Loss of normal lobular architecture but portal triads may be present;

• Usually multifocal;

• Not encapsulated with fibrous tissue;

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Adenoma – Human Vs Rodent

• Humans• Difficult to differentiate from regenerative

hyperplasia

• Usually solitary

• Usually encapsulated

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Carcinoma

Carcinoma: Carcinoma refers to an invasive malignant tumor consisting of transformed epithelial cells. Alternatively, it refers to a malignant tumor composed of transformed cells of unknown histogenesis, but which possess specific molecular or histological characteristics that are associated with epithelial cells, such as the production of cytokeratins or intercellular bridges.

01/05/07

http://en.wikipedia.org/wiki/Malignant

http://en.wikipedia.org/wiki/Tumor

http://en.wikipedia.org/wiki/Epithelium

http://en.wikipedia.org/wiki/Cytokeratins

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Carcinoma Trabecular Type (Malignant)

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Carcinoma Acinar Type (Malignant)

What is this??

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Carcinoma Clear Cell Type (Malignant)

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Carcinoma Scirrhous Type (Malignant)

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Carcinoma Poorly Differentiated (Malignant)

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What is so important about this?

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Carcinoma – Human Vs Rodent

• Humans• Mixed cell tumors are relatively common;• Concurrent cirrhosis is common;• Usually associated with chronic hepatitis;• Rarely spontaneous – usually a history of viral

exposure and/or aflatoxin exposure and/or alcohol exposure.

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Carcinoma – Human Vs Rodent• Rodent

• Classically metastasize to lung (why?)

• Derive from oval cells (pluripotent stem cells) in the periportal area

• Mixed cell tumors (i.e. hepatocyte plus bile duct cell carcinomas) do not occur

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Carcinoma – Human Vs Rodent• Rodent

• Usually do not involve concurrent cirrhosis or chronic hepatitis

• “Spontaneous” in older animals (also in hamsters and beagle dogs)

• “Spontaneous” tumors are common, particularly in some strains.

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So what sort of tumor is this?

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ILSI/HESI MOA Framework

• Is the weight of evidence sufficient to establish the MOA in animals?

• Genotoxic (classically mutagenic)?• Potentially relevant to humans, particularly if

tumors at multiple sites;

• Nongenotoxic (non-mutagenic)?• Relevance to humans is highly dependent on the

mechanism!

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ILSI/HESI MOA Framework• Are the key events in the animal MOA plausible in

humans?

• Genotoxic• Do the mutations occur in human cells in vitro and

in vivo?• Do the same spectrum of mutations occur?• Is the genotoxic progression similar?

• Histopathology• Is the same histopathological life history present in

rodents and humans?

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ILSI/HESI MOA Framework• Are the key events in the animal MOA plausible in humans?

• Nongenotoxic?• Relevance is HIGHLY dependent on the mechanism;

• Do the hyperplastic effect + antiapoptotic effect occur in humans?

• If a receptor-mediated pathway is involved, is this pathway present in humans and of similar pathophysiological relevance?

• Is there a clear dose threshold and what is its relationship to human exposure?

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ILSI/HESI MOA Framework• Taking into account kinetic and dynamic factors, are the key

events in the animal MOA plausible in humans?

• TK is sufficiently similar to result in relevant concentrations at the site of action?

• Promutagens activated to the same extent in humans (i.e. TD issues)? (TD encompasses all mechanisms through which the concentration/amount at the site of action elicits the toxic effect);

• If redox damage is critical, does similar metabolism/events occur in humans?

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• Observation of tumours under different circumstances lends support to the significance of the findings for animal carcinogenicity. Significance is generally increased by the observation of more of the following factors:

• Uncommon tumour types• Tumours at multiple sites • Tumours by more than one route of administration• Tumours in multiple species, strains, or both sexes • Progression of lesions from preneoplastic to benign to

malignant• Reduced latency of neoplastic lesions• Metastases (malignancy, severity of histopath)• Unusual magnitude of tumour response• Proportion of malignant tumours• Dose-related increases• Tumor promulgation following the cessation of exposure

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Relevance Depends on MOA

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Section 4:

Detection and Measurement of Liver Injury

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1. Describe and understand the methods for detection/ measurement/assessment of hepatic toxicity and understand their advantages and limitations.

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Interpretation of Changes in Liver Absolute and Relative Weight.

• Liver weight is strongly correlated with body weight.

• When interpreting changes, it is important to use relative liver weight (i.e. liver to body weight ratios) rather than absolute liver weight

• If you are using absolute liver weights, you must take into account any changes in body weight!

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• Guidance in relation to biological significance of changes in liver weights:

• UK PSD Guidance Document: Interpretation of Liver Enlargement in Regulatory Toxicology Studies 2006 (http://www.pesticides.gov.uk/Resources/CRD/Migrated-Resources/Documents/A/ACP_Paper_on_the_interpretation_of_Liver_Enlargement.pdf)

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• “The toxicological significance of a statistically significant increase in liver weight of ≥ 10% will be interpreted following consideration of the mechanism of action. Findings will be interpreted as potentially adverse, with the specific exceptions of peroxisome proliferators and ‘phenobarbitone-type’ P450 inducers”

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General Aspects of Evaluation of Liver Function.

• Tests of liver function can be used for the following:

– Detect the presence of liver disease.

– Distinguish among different types of liver disorders.

– Gauge the extent of known liver damage

– Follow the response to treatment

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• Limitations common to all tests of liver function:

– Normal results can occur in individuals with serious liver disease (particularly near end-stage disease).

– Liver function tests rarely provide a specific diagnosis; rather they suggest a category of liver disease e.g. hepatocellular or cholestatic.

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• Limitations common to all tests of liver function:

– Functional tests only measure a limited number of hepatic functions (usually only those that are amenable to analysis from blood samples) where as the liver carries out thousands of biochemical functions.

– Many of the common tests do not measure liver function; they most commonly detect cell damage or disruption of bile flow.

– Many of the common tests are influenced by disease outside of the liver i.e. are not absolutely liver specific.

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Classification of Tests of Liver Function.

• Tests based on detoxification and excretory functions:

– Serum bilirubin.

– Urine bilirubin.

– Blood ammonia.

– Serum enzyme levels.

• Tests that detect cellular damage:


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Classification of Tests of Liver Function.

• Tests that measure the biosynthetic function of the liver:

– Serum albumin.

– Coagulation factors.

– Blood ammonia.


• Tests that examine liver function ex vivo.

– Liver slice cultures (experimental only).– 3D tissue cultures– Primary hepatocyte cultures

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Serum Bilirubin Measurement.

• Unconjugated (“indirect”) bilirubin.

– Elevation is rarely due to xenobiotic-induced primary hepatic disease although examples of this effect do exist.

– Mostly associated with diseases/xenobiotics that produce hemolysis. The exceptions are heritable defects of UDP-glucuronyltransferase and impaired bilirubin conjugation (e.g. Gilbert’s syndrome, Crigler-Najjar syndrome).

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– Xenobiotics can produce an increase in serum unconjugated bilirubin without associated hepatic injury if they inhibit bilirubin uptake across the hepatocyte basolateral membrane (flavispidic acid, novobiocin) or inhibit UDP-glucuronyl transferase 1A1 (pregnanediol, chloramphenicol and gemtamicin).

– Remember: in normal adults, the rate limiting step for bilirubin excretion is NOT conjugation by UGT1A1. The rate limiting step is excretion into the bile canaliculi by MRP2! Disruption of the excretion of conjugated bilirubin or leaking back of conjugated bilirubin from damaged bile canaliculi/bile ducts is a far more common xenobiotic injury than disruption of conjugation.

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– As previously discussed the previous point is not true for neonates who have deficient UGA1A1 and are particularly prone to any agent that increases bilirubin production (e.g. hemolytic agents).

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• Conjugated (“direct”) bilirubin.

– Elevated serum conjugated bilirubin almost always implies liver or biliary tract disease.

– Elevation of serum conjugated bilirubin almost always occurs with just about any type of liver disease.

– Prolonged elevations of serum conjugated bilirubin result in covalent rather than reversible binding to albumin which thus delays bilirubin clearance i.e. the decline in serum conjugated bilirubin may be slower than expected following severe or prolonged liver injury.

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• Conjugated (“direct”) bilirubin.– There are at least 2 basic causes of this phenomenon:

• “Leaking back” of conjugated bilirubin from the bile canaliculi or bile ducts due to cholestasis, damage to hepatocytes or bile duct epithelium (loss of tight junctions). This is undoubtedly the most common mechanism.

• Blockage of transport of conjugated bilirubin across the apical hepatocyte membrane (i.e. inhibition of MRP2). THE classical cause of this is glutathione-conjugated sulfobromophthalein which competes for biliary export via MRP2 but this effect occurs with other xenobiotics. Neonates and people with Dubin-Johnson syndrome are particularly prone to these effects since they have relatively low levels of MRP2 on their apical hepatocyte cell membranes.01/05/07

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Urine Bilirubin Measurement.

• Unconjugated bilirubin is always found bound to albumin in serum and thus does not pass through the normal renal glomerulus. Any bilirubin found in urine is almost always conjugated (direct) bilirubin.

• Can be measured very simply using a urine dipstick.

• Theoretically, the urine dipstick test can provide the same information as serum bilirubin measurement, is less invasive and almost 100% accurate.

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Blood Ammonia Measurement.

• Ammonia produced is produced in the body by protein metabolism and by bacteria in the colon. It is detoxified by two routes:

– In the liver by conversion to urea and subsequently excreted by the kidneys.

– In striated muscle where it is conjugated to glutamic acid to produce glutamine.

• Notably, patients with advanced liver disease typically have significant muscle wasting which, in addition to the liver failure, decreases the ability to detoxify ammonia.

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Blood Ammonia Measurement.

• Elevated blood ammonia occurs with:– Advanced liver disease.– Porto-systemic shunting.

• Sometimes used as an indicator of hepatic encephalopathy.

• Problems:– Blood ammonia levels are not correlated with the

presence or severity of hepatic encephalopathy.

– Blood ammonia levels are poorly correlated with hepatic function.

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Blood Enzymes that Reflect Hepatocellular Damage.

• Serum enzyme assays assume that increased serum levels are due to cellular damage, i.e. increased release into the serum, rather than inhibition of enzyme catabolism. Current data suggests that this is a reasonable assumption.

• Serum enzyme levels are insensitive indicators of hepatocellular damage.

• The absolute level of serum enzymes is not a prognostic indicator in hepatocellular injury.

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Blood Enzymes that Reflect Hepatocellular Damage: Alanine Aminotransferase (ALT [SGPT]).

• Primarily found in hepatocytes.

• Normally present in the serum in low concentrations and released in high amounts with hepatocellular damage.

• Looking for a 2-3 times increase for biological significance.

• Level is an indicator of hepatocellular membrane damage rather than hepatocellular necrosis. Serum level of ALT is poorly correlated with the degree of liver cell damage.

• Usually not increased in purely cholestatic disease.01/05/07

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Blood Enzymes that Reflect Hepatocellular Damage: Aspartate Aminotransferase (AST [SGOT]).

• Primarily found in hepatocytes, cardiac muscle, skeletal muscle, kidneys, brain, pancreas, lung, leukocytes and erythrocytes i.e. increased AST in the absence of an increased ALT suggests another source other than liver.

• Looking for a 2-3 times increase for biological significance

• Other features are similar to ALT.

• Level of AST in some species, e.g. horse, is of no meaningful value.

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Blood Enzymes that Reflect Cholestasis: Alkaline Phosphatase (ALP).

• Primarily found in or near the apical hepatocyte membranes (i.e. the canalicular membranes).

• An increase of ALP > 4 times normal is almost always due to cholestasis.

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Blood Enzymes that Reflect Cholestasis: Alkaline Phosphatase (ALP).

• Serum ALP consists of several isoenzymes, each of which is tissue specific (liver, bone, placenta, small intestine). Liver-specific isoenzyme measurement is sometimes required, particularly if significant bone disease is present.

– Heat stability of the different isoenzmes varies: bone and liver ALP are heat sensitive where as placental ALP is heat stable.

– Increases in heat stable ALP strongly suggest placental injury or the presence of an ALP producing tumor.

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Blood Enzymes that Reflect Cholestasis: Gamma Glutamyl Transpeptidase (GGT).

• Located in hepatocyte endoplasmic reticulum and in bile duct epithelial cells.

• Blood levels of this enzyme are considered specific for hepatic disease.

• Because of its diffuse localization in the liver, GGT is considered less specific for cholestasis than ALP.

• Elevated levels of GGT are often interpreted to be evidence of damage to bile duct epithelium.

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Blood Enzymes that Reflect Cholestasis: 5’-nucleotidase.

• Located in or near the apical (i.e. canalicular) hepatocyte cell membrane.

• Rarely elevated in any condition other than cholestasis and therefore considered to be relatively specific.

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Tests Relying on Hepatic Biosynthetic Function:Serum Albumin.

• T1/2 in serum of 15 – 20 days; 1st order kinetics with ~4% degraded per day.

• Because of its long T1/2 and slow turnover, albumin is not a good indicator of acute or mild hepatic dysfunction.

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Tests Relying on Hepatic Biosynthetic Function:Serum Albumin.

• Useful as an indicator of chronic liver disease, particularly cirrhosis where decreases in serum albumin usually reflect decreased albumin synthesis provided other causes of hypoalbuminemia have been ruled out!

• Causes of hypoalbuminemia: malnutrition, protein-loosing enteropathies and nephropathies and chronic infections associated with sustained increases in serum IL-1/TNF (IL-1 and TNF suppress albumin synthesis).

• Albumin measurement is only of clinical value in ~ 0.4% of patients with liver disease!

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Tests Relying on Hepatic Biosynthetic Function:Coagulation Factors.

• With the exception of factor VIII, all functional clotting factors are synthesized by the liver.

• Serum T1/2 for clotting factors ranges from 6 hours (factor VII) to 5 days for fibrinogen.

• The most rapidly depleted clotting factor is factor VII which is critical for the conversion of prothrombin to thrombin during the clotting cascade (thrombin, in turn, converts fibrinogen to fibrin monomer, the basic building block of polymeric fibrin).

• Evidence of coagulopathy that is attributable to liver disease is regarded as a poor prognostic sign.

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Tests Relying on Hepatic Biosynthetic Function:Coagulation Factors.

• The earliest detectable defect is a decline in prothrombin time, followed sometime later by a decline in the activated prothrombin time.

• The decline in PT is associated with the development of clinical evidence of hemorrhage e.g. bruising, ptechial hemorrhages etc.

• Remember, production of active factors II, VII, IX and X require vitamin K i.e. an important differential diagnoses will be vitamin K deficiency, warfarin treatment and anticoagulant rodenticide poisoning.

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Tests Relying on Hepatic Metabolic Clearance:Antipyrine, Caffeine and Galactose Clearance

• More complex to perform and more expensive than conventional biochemical tests, but superior in monitoring the degree of liver dysfunction.

• Involve IV injection of a compound that is mostly or exclusively metabolized by the liver and measuring its clearance from the circulation.

• The antipyrine clearance test is the most common and correlates well with the degree of liver damage.

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Tests Relying on Hepatic Metabolic Clearance:Antipyrine, Caffeine and Galactose Clearance

• The caffeine clearance test is beneficial in severe liver lesions, but practically useless in the case of moderate liver damage.

• The galactose clearance test can be used early in the clinical course of jaundice to distinguish between hepatocellular disease and biliary obstruction.

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Tests That Measure Hepatic Excretion:Sulfobromophthalein (BSP).

• BSP is actively transported across the basolateral hepatocyte membrane by OATP and bilitranslocase.

• BSP is conjugated to glutathione and then transported across the apical hepatocyte membrane by MRP2.

• Competes with conjugated bilirubin for excretion by MRP2. For both bilirubin and sulfobromophthalein, this is the rate limiting step i.e. Like bilirubin, retention of BSP is mostly likely due to competition or inhibition of MRP2 and impaired transport across the hepatocyte apical cell membrane.

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• BSP tests have been largely abandoned in clinical medicine mostly because of cost and complexity.

• They are still extensively used experimentally and are superior to the standard biochemical tests for monitoring the degree of liver dysfunction when significant liver damage is present.

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• Impaired hepatic sulfobromophthalein excretion (i.e. increased or delayed retention) has at least four potential causes:

– Cholestasis due to impaired apical excretion (i.e. inhibition of MRP2). This is the most likely cause since MRP2 function is the rate-limiting step in bilirubin excretion.

– Inhibition of glutathione-S-transferases (requires conjugation to glutathione for excretion).

– Impaired basloateral OATP function.

– Impaired basolateral bilitranslocase function.

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Tests That Measure Hepatic Excretion:Indocyanine Green (ICG).

•ICG is a water-soluble inert compound that is injected intravenously.

• It mainly binds to albumin in the plasma. ICG is then selectively taken up by hepatocytes via the basolateral OAT2 transporter, and subsequently excreted unchanged into the bile via an ATP-dependent transport system.

•ICG is not metabolized; it does not undergo enterohepatic recirculation.

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Tests That Measure Hepatic Excretion:Indocyanine Green (ICG).

•ICG excretion rate in bile reflects the hepatic excretory function and hepatic energy status.

• ICG has been found to be useful to assess liver function in liver donors and transplant recipients, in patients with chronic liver failure and as a prognostic factor in critically ill patients.

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Tests That Measure Hepatic Excretion: Oral Cholecystographic Contrast Agents.

• Technique: a radiocontrast agent that is exclusively excreted vial the biliary system is administered by mouth which allows two forms of observation by CT or MRI:

• Detailed imaging of the biliary tree, particularly the gall bladder.

• Measurement of the time required for the material to appear in the biliary tree and to be completely cleared from the biliary tree (measurements of biliary excretory capacity).

•Common agents used are: iopanoic acid, sodium ipodate, and sodium tyropanoate.01/05/07

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Other Tests.

• Diagnostic imaging

• Ultrasonography, CT and MRI: high sensitivity for detection of biliary duct changes, hepatic masses.

• Doppler CT, doppler ultrasonography and MRI hepatic angiography can be used to assess vasculature and hepatic hemodynamics.

• Endoscopic retrograde cholangiopancreatography (retrograde infiltration of the biliary tract with radiocontrast materials).

• Liver biopsy: remains the gold standard in evaluation of liver disease in living patients.01/05/07

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Section 5.

The Two Basic Classes of Hepatic Toxicants, and Classical “Must Know” Agents Causing Hepatic Damage.

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• Describe and understand the two basic classes of hepatotoxicants and be able to provide examples.

• Describe and intimately understand the mechanisms of classical Class I and Class II hepatotoxins/hepatotoxicants.

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Class I Hepatotoxicants.

• Produce a predictable histologic pattern of hepatic damage in most individuals within a population.

• Severity of damage is dose related.

• Damage can be reliably reproduced experimentally.

• Damage is typically fatty change, necrosis or cholestasis.

• Damage occurs following a brief, but predictable, latent period.

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Classical Examples of Class I Hepatotoxicants.• Acetominophen• Aflatoxin• Allyl alcohol• Bromobenzene

• Carbon tetrachloride (CCl4)

• Chloroform (HCCl3)

• Dimethylnitrosamine• Ethanol• Ethionine• Orotic acid• Phosphorus• Tannic acid• Tetracycline• Thioacetamide• Valproic acid

All produce fatty change or necrosis

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Classical Examples of Class I Hepatotoxicants.

• Carbutamide

• Chlorpropamide

• Chlorpromazine

• Cyclosporine

• Erythromycin

• Lantadene A (from Lantana camara)

• Lithocholic acid.

• α-naphthylisothiocyanate.• Manganese-billirubin.• Methylene dianiline (Epping jaundice)• Methyltestosterone.• Norethandrolone.• Sporodesmin.

All produce cholestasis & bile duct damage

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Class II Hepatotoxicants.

• Non-predictable effects on the liver.

• Idiosyncratic or hypersensitivity/immune-mediated reactions i.e. only affect a small % of the population.

• Severity of lesion is not related to dose.

• Onset of pathology bears no consistent time relationship to exposure.

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Class II Hepatotoxicants.

• Often signs systemic signs of allergic reactions are present (i.e. fever, malaise, arthralgia, eosinophilia, rash).

• Two basic mechanisms:– immune mediated e.g. halothane.– difference in biotransformation (rare genotype).

• Usually not detected by toxicology testing prior to the marketing of a drug.

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Classical Examples of Class II Hepatotoxicants.• Cincophen (antiarrhythmic agent)• Chlorpromazine.• Erthyromycin.• Diclofenac.• Halothane.• Hydrazine MAO inhibitors (iproniazid, iscocarboxazid,

nialamic, isoniazid, phenylzine),• Methyl DOPA.• Indomethacin.• Isoniazid.• Phenylbutazone.• Phenytoin.• Tricrynafen (diuretic used in treatment of heart failure)• Trimethoprim-sulfamethoxazole.• Troglitazone (anti-diabetic drug).01/05/07

Must Know Class I Hepatotoxicants: Acetominophen.

• Mechanism:

AcetominophenCYP2E1

N-acetyl-p-benzoquinone imine(nucleophile)

Sulfonation

Detoxified if Low Dose

Conjugation to GSH

Mercapturic acid

Centrilobular Necrosis

Long-term alcohol abuse has been established as potentiating acetaminophen toxicity via induction of CYP2E1 and depletion of glutathione. Alcoholic patients may develop severe, even fatal, toxic liver injury after ingestion of standard therapeutic doses of acetaminophen.

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Must Know Class I Hepatotoxicants: Acetominophen.

• How would the following factors affect acetominophen toxicity?– Prior exposure to isopropyl alcohol?– Prior exposure to ethanol?– Prior treatment with phenobarbital?

– Prior exposure to CCl4

– Concurrent diethylmaleate treatment (depletes GSH)?– Concurrent treatment with piperonyl butoxide (inhibits

CYP2E1)?– Concurrent treatment with SKF 525a?– Exposure in cats?

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Must Know Class I Hepatotoxicants: Aflatoxin.

• Mycotoxin produced on stored grains by Aspergillus flavus and A. parasiticus on cereal grains.

• Acute exposure results in centrilobular necrosis; hepatic carcinogen in some species (including humans) with chronic exposure.

• Humans are particularly resistant to acute hepatic injury by aflatoxins; growth retardation in children and hepatic carcinoma are THE major problems in humans.

• Mechanism of acute hepatic injury (Periportal/Zone 1):

Aflatoxin B1

CYP2E1 Aflatoxin epoxide (Aflatoxin M

1 )

Why is damage in Zone 1?01/05/07

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Must Know Class I Hepatotoxicants: Allyl Alcohol.

• Important chemical precursor and common byproduct of combustion of organic materials (including fuels).

• Produces periportal necrosis.• Allyl alcohol is a metabolite of cyclophosphamide and is

responsible for some of this drug's effects.• Mechanism:

Allyl alcohol ADH

Acrolein

NAD+ NADH

Lipid peroxidationDepletion ofGSHProtein damage

Why do you think that the hepatic damage due to allyl alcohol occurs in zone I (perioportal area)?

Allyl alcohol is a suicide substrate: what do you think that prior exposure would have on a second treatment with allyl alcohol?

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Must Know Class I Hepatotoxicants: Amanita phalloides (Death Cap Mushroom).

• Toxins are amatoxins and phallotoxins (both are cyclopeptides).

• Amatoxins have a propensity to concentrate in hepatocytes because of active uptake by OAT1B3.

– Competitive substrates for OAT1B3, such as rifampicin, have been theoretically suggested for treatment

• Amatoxins inhibit RNA polymerase II, therefore interfering with DNA and RNA transcription

• Phallotoxins interrupt the actin polymerization-depolymerization cycle and thus may contribute to the liver disease due to suppression of bile canalicular motility.

• Effect is centrilobular necrosis.

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Must Know Class I Hepatotoxicants: Bromobenzene (Phenylbromide).

• ONE OF THE CLASSICAL EXAMPLES OF CYP2E1 TOXICATION

• Mechanism:

CYP

2E1

Epoxide hydrolase

Centrilobular/Z3 necrosis

Toxicity inhibited by inhibitors of CYP2E1

Toxicity enhanced by inducers of CYP2E1

Toxicity enhanced by depletors of GSH

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Must Know Class I Hepatotoxicants: Carbon Tetrachloride.

• Another of THE classical examples of CYP2E1 toxication• Mechanism:

CYP2E1

Trichloromeithyl radical acts to: Reacts with hydrogen to form chloroform which is then metabolized to

a radical Reacts with itself to form hexachloroethane Reacts with proteins -> SUICIDE SUBSTRATE FOR CYP2E1 Peroxidizes the polyenoic lipids of the endoplasmic reticulum and

triggers the subsequent generation of secondary free radicals derived from the lipids in the membrane --> destroys the endoplasmic reticulum resulting in decreased CYP activity and decreased protein synthesis

Triggers fatty liver by blocking the binding of triglycerol to apoproteins thus blocking excretion of apolipoproteins from hepatocytes01/05/07

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Must Know Class I Hepatotoxicants: Carbon Tetrachloride.

• Classical effect is centrilobular/zone 3 fatty change/necrosis

• CCl4 is the best studied example of the effects of modulation

of CYP levels and tissue damage.

• Potentiators of CCl4 hepatotoxicity:

– Prior exposure to any CYP2E1 inducer e.g. Ethanol, most ketones (acetone), diabetes mellitus, isopropyl alcohol (converted to acetone by ADH), phenobarbital

• Inhibitors of CCl4 hepatotoxicity:

– Piperonyl butoxide inhibition of CYP2E1 (remember: initially inhibits CYP but then later induces it! Effect depends on timing!)

– SKF525a inhibition of CYP2E1

– Concurrent treatment with a CYP2E1 substrate e.g. concurrent treatment with ethanol, acetone

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Must Know Class I Hepatotoxicants: Chloroform.

• Classical effect is centrilobular/zone 3 fatty change/necrosis• Mechanism:NU = tissue nuleophile

Phosgene

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Must Know Class I Hepatotoxicants: Chloroform.

• Classical effect is centrilobular/zone 3 fatty change/necrosis: WHY?

• Common source of human exposure is chlorinated drinking water.

• Modulation of toxicity by modulation of CYP2E1 resembles that of CCl

4.

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Must Know Class I Hepatotoxicants: Copper.

• Several distinct diseases that all involve damage to the liver

– Acute copper poisoning.

– Wilson's disease, the Long-Evans cinnamon rat and toxic milk mice.

– Idiopathic childhood cirrhosis and copper storage disease in Bedlington Terriers.

– Copper toxicity in ruminants.

– Copper hepatotoxicity secondary to cholestatic defects: Tyrolean childhood cirrhosis, Indian childhood cirrhosis, North Ronaldsay sheep, Doberman Pinscher hepatitis, Sky Terrier hepatitis, & non-suppurative feline cholangioheptatitis complex.

• All require a little bit of knowledge about Cu metabolism, storage and excretion (next couple of slides).

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GUT ENTEROCYTE

Cu2+

S2-

CuS

Chelate

S2-+ MoO42-

Terththiomolybdate

hCTR1

DMT1

?

Cu2+

ATB7A Cu2+

Storage?

Cu-AlbuminCu-RBC

Cu-Histidine

AlbuminRBCsHistidine

CIRCULATION

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hCTR1

Cu-Albumin

Albumin

Cu2+

Space of Disse

MoO42- + protein

CuM

oO4 -protein com

plexBileHepatocyte

Cu-metallothionine

Cu-Atox1

Lysosome

ATP7b Cu2+

Cu2+Murr1

Murr-1-linked endosome

Cu2+

Exocytosis

Apoceruloplasmin

CeruloplasminCeruloplasmin

ATP7b is defective in Wilson's Disease, LEC rat & toxic milk mice; Murr1 is defective in Bedlington terriers and idiopathic childhood cirrhosis

Ruminants in particular

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Must Know Class I Hepatotoxicants: Acute Copper Toxicity.

• Remember: the primary target is the gut. Liver, hematlogical and kidney disease will occur in those that survive the initial GI syndrome.

• Produces centrilobular hepatic necrosis.

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Must Know Class I Hepatotoxicants: Wilson's Disease, LEC Rat, Toxic Milk Mice.

• Humans with Wilson's disease, LEC rats and toxic milk mice lack ATP7b and cannot synthesize ceruloplasmin and thus accumulate copper in the liver, cornea and CNS.

• Untreated Wilson's disease is associated with chronic active centrilobular hepatitis and eventual cirrhosis due to hepatic copper accumulation, damage to the cornea and significant neuropsychiatric disease.

• Treatment is by provision of a low copper diet, the use of copper chelators such as tetramine or penicillamine, inclusion of ammonium tetrathiomolybdate in the diet and increased dietary Zn, which competes with copper for GI absorption.

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Must Know Class I Hepatotoxicants: Idiopathic Childhood Cirrhosis and Copper Storage Disease in Bedlington Terriers.

• Both diseases are due to a defect in Murr1 which prevents the exocytosis of endosomal copper into the bile.

• The diseases are characterized by centrilobular chronic active hepatitis, hepatic fibrosis and eventual death if untreated

• In idiopathic childhood cirrhosis, storage of food items in copper utensils, particularly the storage of acidic materials like milk in copper containers.

• Unlike Wilson's disease, Kayser-Fleischer lines, renal and neuropsychiatric disease does not occur in ICC or the copper storage disease in Bedlington Terriers.

• Treatment is similar to Wilson's disease.

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Must Know Class I Hepatotoxicants: Copper Toxicity in Ruminants.

• Disease is a cause of major livestock losses, particularly in sheep.

• Disease is caused by a relative imbalance in the amounts of copper and molybdenum in the diet and the disease is better termed “chronic copper excess/molybdenum deficiency.”

• Disease is exactly the same as molybdenum deficiency.• Cattle, goats, swine, dogs, chickens and turkeys are

relatively resistant to this problem. The problem has never been recorded in horses.

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• Causes and factors affecting the disease:

– Consumption of any diet (grazed or compounded) with a Cu:Mo ratio > 10:1.

– Many grazing areas in the Midwest, the Great Plains and Central Canada contain sufficient levels of copper and low enough levels of molybdenum to make the GRAS addition of copper to stock feeds at the usual rate of 15 ppm potentially toxic.

– Improved pastures containing large amounts of Trifolium subterraneum (subterranean clover): contains little or no Mo.

– Consumption of pastures contaminated with copper containing pesticides/fungicides (particularly near orchards).

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• Causes and factors affecting the disease:

– Anything that impairs liver function (even when diets containing safe levels of Cu are fed) may ↓ liver Cu metabolism and excretion, ↑ liver Cu accumulation and predispose to chronic Cu toxicity.

– Pyrrolizidine alkaloids are particularly important in Australia/New Zealand.

• Heliotropium sp, Echium sp (particularly E. plantagineum; Paterson’s curse), Senecio sp

Lupine alkaloids

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Pathogenesis Essentially a 2 phase disease: Phase I: Characterized by the absence of disease and

chronic hepatic Cu accumulation (weeks to months). Phase II: Clinical disease phase characterized by

hemolytic crisis, renal failure and liver damage.

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Phase I: chronic hepatic Cu accumulation (weeks to months)

Disease is completely subclinical at this phase, although it is detectable using specialized histology (see next few slides)

Characterized by progressive lysosomal accumulation of copper in the liver

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Excessive copper accumulation in hepatocytes in ovine copper toxicity (rhodanine stain for copper); excessive copper is also usually present in large amounts in hepatic Kupffer cells. Copper can also be identified using the rubeanic acid stain. REMEMBER: LARGE AMOUNTS OF COPPER WILL ONLY BE PRESENT IN THE LIVERS OF ASYMPTOMATIC

ANIMALS (I.E. BEFORE THE ACUTE HEMOLYTIC PHASE)!01/05/07

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Excessive hepatocyte coper in ovine copper toxicity (Victoria blue stain: stains copper-associated protein); excessive copper is also usually present in large

amounts in hepatic Kupffer cells. REMEMBER: LARGE AMOUNTS OF COPPER WILL ONLY BE PRESENT IN THE LIVERS OF ASYMPTOMATIC ANIMALS (I.E.

BEFORE THE ACUTE HEMOLYTIC PHASE)!01/05/07

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Phase II: “Acute disease phase.” Cu levels reach a crisis point beyond which the liver cannot

excrete sufficient copper or store it in a safe manner ± other triggering factors hepatocellular death immature replacement hepatocytes are unable to rapidly absorb and clear the excess Cu sudden release of large amounts of Cu into the circulation oxidative erythrocyte cell membrane damage and oxidation of hemoglobin to methemoglobin intravascular hemolysis and methemoglobinemia ↓ blood O2 carrying capacity centrilobular hepatic anoxia/necrosis further Cu release.

Triggers include: hepatic toxins, reduced food intake, handling, strenuous exercise, sudden intake of Cu containing foods, sudden cold weather or any other stressor.

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Pale, swollen friable livers associated with the acute phase of copper toxicity in sheep.

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Liver: Necrosis, centrilobular to submassive, with hemorrhage due to copper toxicity in sheep

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Classical “Gunmetal Blue” kidneys from sheep with copper toxicity

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Must Know Class I Hepatotoxicants: Copper Toxicity Secondary to Cholestatic Defects.

Importantly, hepatic copper accumulation an associated hepatic disease can occur secondary to just about any chronic cholestatic condition, be it toxicant-induced or genetic.

Classical examples of this phenomenon, all of which combine a genetic cholestatic defect with environmental copper association are Humans: Tyrolean childhood cirrhosis, Indian childhood cirrhosis. Domestic animals: North Ronaldsay sheep, Doberman Pinscher

hepatitis, Sky Terrier hepatitis, & non-suppurative feline cholangioheptatitis complex.

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Must Know Class I Hepatotoxicants: Cyclophosphamide.

• Cancer chemotherapeutic; side-effect is severe liver damage.

• Target is the liver sinusoids.

• Toxicity is due to metabolism to acrolein and phosphoramide mustard:

260

Must Know Class I Hepatotoxicants: Endotoxin.

• Classical target is Kupffer cells due to selective accumulation.• Endotoxins (e.g. LPS) trigger Kupffer cell activation and the

release of cytokines and reactive oxygen species which, in turn, trigger inflammation and extensive parenchymal damage.

• Endotoxins and Kupffer cells appear to play a key role in ethnol-induced chronic liver disease:

– Ethanol exposure results in increased endotoxin release and uptake by Kupffer cells.

– Endotoxin exposure appears to “prime” the liver for damage by ethanol.

– Endotoxin is an inducer of ADH and enhances free radical production associated with ethanol metabolism

– Endotoxin depletes GSH content in hepatocytes, reducing the detoxification of free radicals

– Endotoxin stimulates the laying down of collagen by Ito cells, thus favoring inappropriate repair over regeneration

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Must Know Class I Hepatotoxicants: Ethanol.• Without question THE MAJOR CAUSE of toxic liver disease in

humans.• In almost all cases, ethanol consumption makes all other

forms of liver disease (toxic or otherwise) worse.

Predominates in non-addicts

Important in addicts

Important in addicts

ADH occurs in 3 isoforms in humans: ADH1, ADH2, ADH3

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Must Know Class I Hepatotoxicants: Ethanol Fatty Liver.

• Pathogenesis of alcohol-induced fatty liver:

– Can occur acutely after consumption of surprisingly low amounts of ethanol over a surprisingly short period! 90-100% of patients with alcohol hepatitis will also have alcohol-induced fatty liver.

– There are 3 main theories regarding the pathophysiology of ethanol-induced fatty liver:

• Decreased NAD/NADH ratio theory• Modulation of the hypothalamic-pituitary-adrenal axis

by ethanol consumption theory. • Inhibition of the release of VLDL into the circulation

theory.

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• Pathogenesis of alcohol-induced fatty liver:

– Decreased NAD/NADH ratio theory• Metabolism of EtOh results in reduced amounts of

NAD+ in hepatocytes. This, in turn, is associated with inhibition of glycerol-3-phosphate dehydrogenase (NAD dependent), glycolysis and gluconeogenesis. Cellular accumulation of glycerol-3-phosphate ensues which results in enhanced esterification of fatty acids to form triacylglycerols (neutral fats) that accumulate in hepatocytes and a shift in metabolism towards ketogenesis.

• Decreased NAD/NADH ratio results in decreased availability of NAD+ for β-oxidation of fatty acids.

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• Pathogenesis of acute alcohol fatty liver:

– Modulation of the hypothalamic-pituitary-adrenal axis by ethanol consumption theory.

• GI irritation by ethanol results in the release of arginine vasopressin → stimulates release of ACTH due to activation of the V1b receptor in the anterior pituitary → ↑ cortisol → increased lipid mobilization → accumulation of fatty acids within the hepatocyte at a rate that exceeds the capacity for β-oxidation.

– Inhibition of the release of VLDL into the circulation theory.• Chronic ethanol consumption results in inhibition of

apolipoprotein B synthesis and decreased VLDL synthesis and secretion by hepatocytes.

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• Centrilobular localization of steatosis results from decreased energy stores from relative hypoxia and a shift in lipid metabolism, along with a shift in the redox reaction caused by the preferential oxidation of alcohol in the central zone.

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• Consequences of alcohol-induced fatty liver:

– Increased collagen turnover within the liver.

– increased propensity for cirrhosis and other fibrotic liver diseases.

– Associated with an increased propensity for liver cancer in humans.

– Large changes in drug metabolism by the liver, particularly due to induction of CYP2E1

– Large changes in hormone catabolism by the liver.

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Must Know Class I Hepatotoxicants: Ethanol Hepatitis.

• Pathogenesis of alcohol hepatitis.

– There are several theories regarding the pathophysiology:• Protein-energy malnutrition theory.• Alterations in cell membranes theory.• Indution of an altered metabolic state in hepatocytes

theory.• Generation of free radicals and oxidative injury theory.• Acetaldehyde-associated damage theory.• The endotoxin-cytokine-Kupffer cell activation, cytokine

release and inflammation theory.• Induction of autoimmune hepatitis theory.• Exacerbation of hepatitis viral infections.

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• Protein-energy malnutrition theory.

– Most patients with alcoholic hepatitis exhibit evidence of protein-energy malnutrition (PEM). In the past, nutritional deficiencies were assumed to play a major role in the development of liver injury. This assumption was supported by several animal models in which susceptibility to alcohol-induced cirrhosis could be produced by diets deficient in choline and methionine.

– This view changed in the early 1970s after key studies by Lieber and DiCarlo performed in baboons demonstrated that alcohol ingestion could lead to steatohepatitis and cirrhosis in the presence of a nutritionally complete diet.

– However, recent studies suggest that enteral or parenteral nutritional supplementation in patients with alcoholic hepatitis may improve survival.

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Must Know Class I Hepatotoxicants:Ethanol Hepatitis.

• Altered cell membrane theory.

– Ethanol and its metabolite, acetaldehyde, have been shown to damage liver cell membranes.

– Ethanol can alter the fluidity of cell membranes, thereby altering the activity of membrane-bound enzymes and transport proteins.

– Ethanol damage to mitochondrial membranes may be responsible for the giant mitochondria (megamitochondria) observed in patients with alcoholic hepatitis.

– Acetaldehyde-modified proteins and lipids on the cell surface may behave as neoantigens and trigger immunologic injury.

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• Indution of an altered metabolic state in hepatocytes theory.

– Hepatic injury in alcoholic hepatitis is most prominent in the centrilobular area (zone 3) of the hepatic lobule. This zone is known to be the most dependent on anerobic metabolism.

– The reduced NAD/NADH ratio that occurs in hepatocytes exposed to ethanol results in inhibition of the energy-producing steps in glycolysis. The net result is decreased anaerobic generation of ATP in the area of the liver lobule that is most dependent on anerobic metabolism (i.e. Zone 3).

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– Chronic alcohol consumption depresses the activity of all mitochondrial complexes, except complex II.

– Several abnormalities in mitochondrial respiratory chain have been described:

• Decreased activity and heme content of cytochrome oxidase.

• Impaired electron transport and proton translocation through complex I.

• Cecreased cytochrome b content in complex III.• Reduced function in ATP synthase complex.

– The net result is severe impairment of mitochodrial generation of ATP via oxidative phosphorylation.

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– Ethanol induces a hypermetabolic state in the hepatocytes, partially because ethanol metabolism via CYP2E1 does not result in energy capture via formation of ATP via ethanol metabolism. Rather, this pathway leads to loss of energy in the form of excessive heat production.

– Decreased NAD/NADH ratio also results in the inhibition of gluconeogenesis. and inhibition of energy generation by β-oxidation of fatty acids.

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• Generation of free radicals and oxidative injury theory.

– Due to the decreased NAD/NADH ratio, there is an increased availability of reducing equivalents, such as NADH, which leads to their shunting into mitochondria, which induces the electron transport chain components to assume a reduced state. This facilitates the transfer of an electron to molecular oxygen to generate reactive species as superoxide anion.

– Mitochondrial ROS generation can also derive from the ethanol-induced changes in the mitochondrial respiratory chain. These changes promote superoxide anion generation within the mitochondria which leads to cell damage and necrosis.

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– Free radicals, superoxide and hydroperoxides, are generated as byproducts of ethanol metabolism via CYP2E1 and catalase pathways which become predominant in chronic alcoholism.

• CYP2E1 interacts with cytochrome reductase, which leads to electron leaks in the respiratory chain and ROS production. The species produced in this cascade can interact with iron (Fenton reaction) generating even more potent hydroxyl, ferryl and perferryl radicals which perpetuate liver damage

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– Acetaldehyde reacts with glutathione and depletes this key element of the hepatocytic defense against free radicals.

– Other antioxidant defenses, including selenium, zinc, and vitamin E, are often reduced in individuals with alcoholism, possibly due to malnutrition.

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• Acetaldehyde associated damage theory.

– Levels of acetaldehyde in the liver represent a balance between its rate of formation (determined by the alcohol load and activities of the 3 alcohol-dehydrogenating enzymes) and its rate of degradation by ALDH. ALDH is down-regulated by long-term ethanol abuse, with resultant acetaldehyde accumulation.

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• Acetaldehyde associated damage theory.

– The deleterious effects of acetaldehyde accumulation in hepatocytes include:

• Impaired β-oxidation of fatty acids → fatty liver and impaired energy metabolism

• Acetaldehyde covalently binds with hepatic macromolecules, such as amines and thiols, in cell membranes, enzymes, and microtubules to form acetaldehyde adducts. This binding may trigger an immune response through formation of neoantigens, impair function of intracellular transport through precipitation of intermediate filaments and other cytoskeletal elements, and stimulate Ito cells to produce collagen.

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Must Know Class I Hepatotoxicants: Ethanol Hepatitis.• The endotoxin-cytokine-Kupffer cell activation, cytokine

release and inflammation theory.

Increased endotoxin uptake from gut

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• Alcohol-induced autoimmune hepatitis theory.

– Active alcoholic hepatitis often persists for months after cessation of drinking: in fact, its severity may worsen during the first few weeks of abstinence. This observation suggests that an immunologic mechanism may be responsible for perpetuation of the injury.

– Levels of serum immunoglobulins, especially the immunoglobulin A class, are increased in persons with alcoholic hepatitis.

– Antibodies directed against acetaldehyde-modified cytoskeletal proteins can be demonstrated in some individuals.

– Autoantibodies, including antinuclear and anti–single-stranded or anti–double-stranded DNA antibodies, have also been detected in some patients with alcoholic liver disease.

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Must Know Class I Hepatotoxicants: Methylene Dianiline.

• Famous because of “Epping Jaundice”: an outbreak of acute cholestatic jaundice in the English city of Epping due to consumption of bread that became contaminated with methylene dianiline during transportation.

• MD specifically targets bile duct epithelial cells causing acute cholestatsis.

• Mechanism is uncertain:

– Phase I N-acetylation of MD by both NAT1 and NAT2 enhances the disease and individuals that have the fast N-acetylating NAT2 genotype are more susceptible to the poisoning.

– Glutathione depletion enhances the disease, implying an oxidative or free-radical-dependent mechanism

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Normal rat portal triad for comparison with the next slide. Note the healthy bile duct epithelium.01/05/07

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Portal triad of a rat treated with methylene dianiline. Note that the bile ducts (marked with “*”) have lost their epithelium and the inflammatory infiltrate (marked by the “►”).01/05/07

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Must Know Class I Hepatotoxicants: Microcystins and Nodularins.

• Cyanobacterial toxins associated with cyanobacterial blooms in water (classically by Microcystis aeruginosa and Nodularia spumigena,, but they are also produced by other cyanobacterial species; classically, microcystin-LR is most associated with Microcystis cyanobacterial blooms).

• Cyclic peptides.

• Concentrate in the liver due to active uptake by OATP

• Inhibit protein phosphatases 1 and 2A which results in this leads to the rapid disaggregation of intermediate filaments (cytokeratins) that form the cellular scaffold. Microfilaments become detached from the cytoplasmic membrane, which results in cell cytoskeletal deformation and bleb formation. Cell lysis and apoptosis follow, depending on dose.

• Unusually, the necrosis is primarily midzonal (zone 2).

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Must Know Class I Hepatotoxicants: Pyrrolizidine Alkaloids.

• Probably THE most important of the plant hepatotoxicants.• On a world-wide basis, cause $billions of losses to the animal industries.• Major epidemics of PA poisoning in humans have occurred.• PAs are notable food contaminants, particularly in honey from hives grazed

on PA containing plants, and in grains contaminated with the seeds from PA producing plants.

• The principal families involved are the Asteraceae (Compositae), Boraginaceae and Leguminaceae (Fabaceae), while the main genera are Senecio (Asteraceae), Crotalaria (Leguminaceae), Heliotroprium, Echium, Trichodesma, and Symphytum (Boraginaceae).

• The most famous plant involved in PA poisoning of livestock in Oregon is Tansy Ragwort (Senecio jacobaea).

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Dr R B Cope 286– Tansy Ragwort (Senecio jacobaea).01/05/07

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• PAs are metabolized within hepatocytes to a reactive pyrrole which react with cellular macromolecules at or near the site of formation. – They bind most strongly with sulphydryl groups but also with amino

groups of proteins and nucleic acid bases.• Many of the reactive pyrroles have a sufficiently long half-life to allow for

damage to the structures surrounding the hepatocytes. The sinusoidal endothelium is particularly sensitive.– In the case of the PAs from Crotalaria spectabilis, the reactive pyrroles

are long lived and are carried by RBCs to the lung where they induce damage to the pulmonary vasculature

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• PA-induced hepatic disease usually takes 2 forms:– Acute hepatic disease characterized by centrilobular necrosis and acute

hepatic failure.– More commonly: chronic liver disease characterized by cirrhosis, veno-

occlusive disease (“peliosis hepatis”) and attempts at hepatic regeneration.

– The development of abnormal hepatocyte megalocytes is is a characteristic feature of the liver pathology. PAs cause alkylation of DNA which impairs the proliferation of endogenous hepatocytes which results in hepatocytes that enlarge (megalocytes) but cannot complete cell division. The net result is that hepatic regeneration is ineffective.

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Must Know Class I Hepatotoxicants: Sporodesmin.

• Mycotoxin produced by Pithomyces chartarum that grows on perennial rye grass (Lolium perenne)

• Major disease of ruminants grazed on perennial rye grass pastures.

• Concentrates in the bile and undergoes futile redox cycling resulting in free radical damage to the canalicular hepatocyte cell membrane. Net result is cholangiohepatitis and secondary photosensitization due to phylloerythrin accumulation (facial eczema in sheep).

• Redox cycling of sporodesmin is strongly catalyzed by copper and agents that reduce copper absorption (e.g. Zinc) reduce the toxicity.

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Must Know Class II Hepatotoxicants: Halothane.

• Halothane is probably THE best studied human Type II hepatotoxicant.

• There are two types of halothane toxicity:– Type I: predominantes in rodents and is usually very

mild in humans. This is due to the formation of a reactive metabolite.

– Type II: only occurs in humans and is very severe. This is an autoimmune hepatitis due to neoantigen formation.

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Must Know Class II Hepatotoxicants: Halothane.

Trifluoroacetylchloride Binds to protein

Neoantigen formation

Autoimmune hepatitis in humans

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Must Know Class II Hepatotoxicants: Diclofenac.

• Diclofenac is a NSAID. Similar forms of drug-induced autoimmune hepatitis occur with many NSAIDs.

• NSAIDs act as both Type I and Type II hepatotoxicants– Type I mechanism: due to dysregulation of hepatocyte

mitochondrial function and futile REDOX cycling– Type II mechanism: diclofenac metabolites form

protein adducts within the hepatocyte resulting in neoantigen formation and immune-mediated hepatitis.

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Example of Unexpected Drug-Induced Liver Failure Detected During Post-Market Surveillance.

FDA Public Health Advisory

Ketek (telithromycin) Tablets

(Currently being updated)

Today, January 20, 2006, Annals of Internal Medicine published an article reporting three patients who experienced serious liver toxicity following administration of Ketek (telithromycin). These cases have also been reported to FDA MedWatch. Telithromycin is marketed and used extensively in many other countries, including countries in Europe and Japan. While it is difficult to determine the actual frequency of adverse events from voluntary reporting systems such as the MedWatch program, the FDA is continuing to evaluate the issue of liver problems in association with use of telithromycin in order to determine if labeling changes or other actions are warranted. As a part of this, FDA is continuing to work to understand better the frequency of liver-related adverse events reported for approved antibiotics, including telithromycin. While FDA is continuing its investigation of this issue, we are providing the following recommendations to healthcare providers and patients:

Healthcare providers should monitor patients taking telithromycin for signs or symptoms of liver problems. Telithromycin should be stopped in patients who develop signs or symptoms of liver problems.

Patients who have been prescribed telithromycin and are not experiencing side effects such as jaundice should continue taking their medicine as prescribed unless otherwise directed by their healthcare provider.

Patients who notice any yellowing of their eyes or skin or other problems like blurry vision should contact their healthcare provider immediately.

As with all antibiotics, telithromycin should only be used for infections caused by a susceptible microorganism. Telithromycin is not effective in treating viral infections, so a patient with a viral infection should not receive telithromycin since they would be exposed to the risk of side effects without any benefit.

The case review in today’s online publication by Annals of Internal Medicine reports three serious adverse events following administration of telithromycin. All three patients developed jaundice and abnormal liver function. One patient recovered, one required a transplant, and one died. When the livers of the latter two patients were examined in the laboratory, they showed massive tissue death. These two patients had reported some alcohol use. All three patients had previously been healthy and were not using other prescription drugs. The FDA is also aware that these patients were all treated by physicians in the same geographic area. The significance of this observation is not clear at the present time.

In pre-marketing clinical studies, including a large safety trial and data from other countries, the occurrence of liver problems was infrequent and usually reversible. Based on the pre-marketing clinical data, it appeared that the risk of liver injury with telithromycin was similar to that of other marketed antibiotics. Nonetheless, the product label advises doctors about the potential for liver-related adverse events associated with the use of telithromycin.

Telithromycin is an antibiotic of the ketolide class. It was the first antibiotic of this class to be approved by the FDA in April, 2004 for the treatment of respiratory infections in adults caused by several types of susceptible microorganisms including Streptococcus pneumoniae and Haemophilus influenzae.01/05/07

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Example of Unexpected Drug-Induced Liver Failure Detected During Post-Market Surveillance.

Approx 50% of telithromycin is metabolized to an inactive metabolite by CYP3A4.

Question: is this yet another case of phase I toxication or is another mechanism involved?

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Examples of Unexpected Drug-Induced Liver Failure Detected During Post-Market Surveillance.

• Other recent examples (2006 and 2007 )of unexpected drug-induced liver toxicity that was detected by post-market surveillance:• Ketek (telithromycin)

• Cymbalta (duloxetine hydrochloride)

• Betaseron (interferon beta-1b)

• Viramune (nevirapine)

• Serzone (nefazodone hydrochloride)

• Kava kava (“natural” health supplement i.e. regulated as a food and not a drug).

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Unexpected Drug-Induced Liver Failure Detected During Post-Market Surveillance.

• Why so many?• Idiosyncratic reactions:

• Currently there is no possible way to test a large enough number of animals or humans to ensure that every genotype is examined.

• Use of inbred (i.e. syngenomic) strains in pre-market testing.

• Economic pressure: tendency to ignore or “weasel word” a way around the one or two patients that have severe toxic reactions during the clinical trials (as was the case with telithromycin) due to the high costs of bringing a new drug to market (total development costs from concept to market is now approaching $US1 billion and 10 years of work for each new molecule!)

• Toxicogenomics has reduced this problem, but it is not perfect: not every single allele has been sequenced let alone incorporated onto a gene chip.

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The Latest Lawsuit Due to Unexpected Drug-Induced Liver Failure Detected During Post-Market Surveillance.

• Why so many?

• Immune-mediated reactions: • Current techniques are good at detecting strong

sensitizers but are very poor at detecting marginal or weak sensitizers that sometimes take months or years of exposure before the immune reaction is manifested.

• There is currently no way to test every Ig idiotype or T-cell receptor type present in the entire human population for reactivity with a new drug (although there are some obvious guidelines e.g. avoid molecules or metabolites that have covalent binding to host proteins i.e. behave like hapten-carriers).

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Section 6:

Mode of Action of Rodent Forestomach Tumours:Relevance to Humans.

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1. Under the ILSI/HESI mode of action framework for interpretation of stomach tumour data for human risk assessment.

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Gross anatomy of murine forestomach after NMBA (N-nitrosomethylbenzylamine) treatment.

Zanesi N et al. PNAS 2001;98:10250-10255

©2001 by The National Academy of Sciences

NMBA-induced histopathology of murine forestomach.

Zanesi N et al. PNAS 2001;98:10250-10255

©2001 by The National Academy of Sciences

Introduction

• Forestomach tumors/pre-neoplastic lesions in rats and mice are a common finding in repeat-dose toxicology studies;

• Debate over the human relevance due to:• Dose and exposure differences between rodents and

humans; • Substantial toxicokinetic differences (exposure);• Substantial anatomical differences;• Substantial physiological/metabolic differences of the

forestomach epithelium;• Different mechanisms and tumor types in humans

compared with rodents;

Dose and Exposure Problems• Doses used in rodent oral carcinogenesis often far exceed

normal human environmental exposure conditions (possible rare exception is some direct food additives);

• Doses that produce forestomach irritation in rodents really should be considered as exceeding the MTD – i.e. poor practice in rodent carcinogenesis studies and not according to GLP/test guidelines;

Dose and Exposure Problems

• Gavage can produce forestomach irritation and is not physiological:– Large volumes;– Damage to the mucosa;– Esophageal reflux;– Possibly replicates tablets (but not capsules);

Tissue specificity• Forestomach carcinogens divisible into at least 3

categories:

– Produce forestomach tumors and tumors at other sites when administered by gavage;

– Produce only forestomach tumors when administered by gavage;

– Produce forestomach tumors and tumors when administered by non-oral routes;

• In terms of human relevance, forestomach + tumors at other sites is likely to be more important except in the case of site of first contact carcinogens.

Tissue concordance/anatomical issues• Humans do not have a forestomach or a pars esophagea:

– Roughly equivalent tissue in terms of histology is the esophagus;

– Humans do not store food in the esophagus where as rodents store food in the forestomach;

– Transit time through the human stomach is lower than transit time through the rodent stomach (forestomach) difference in tissue exposure;

– Chemicals pass quickly through the human esophagus and thus the exposure is very limited compared with chemical exposure of the rodent forestomach.

Tissue concordance/anatomical issues

• Physiological issues:

– Rodent forestomach does not have a protective mucous coating increased tissue exposure to chemicals and more prone to irritant effects;

– pH in rodent forestomach is higher than the pH of the human stomach relevant to detoxification (e.g. hexavalent chromium to trivalent chromium in low pH of human stomach);

– Potential metabolic differences of rodent forestomach epithelium conversion of 2-butoxy ethanol to 2-butoxyacetic acid in rodent forestomach but not in human stomach;

Tumour types and biology issues• Rodents

– Predominant tumor types are papillomas (non-malignant) and squamous cell (low malignancy – regional metastasis) carcinomas;

– Typically located at the limiting ridge;

– Possibly have some relevance to human esophageal squamous cell carcinoma BUT chemical exposure of the human esophagus is much lower than in the rodent forestomach due to much lower transit time (no storage in esophagus);

– Not relevant to human esophageal adenocarcinoma.

Tumour Types and Biology Issues

• Humans

– All human stomach cancers are gastric adenocarcinomas and arise from the glandular epithelium;

– Rodent forestomach tumors have a different histiogenesis and are not relevant to the human gastric tumors;

Genotoxicity Issues• Forestomach carcinogens are divisible into 2 basic groups:

– DNA reactive chemicals (classical in vivo genotoxic carcinogens)

• Site of first contact carcinogens (generally direct acting carcinogens and are usually highly reactive chemicals; typically direct acting alkylating agents);

• Classical pro-carcinogen DNA reactive chemicals;

– Non-DNA reactive chemicals (classical non-genotoxic carcinogens);

• Typically irritant chemicals or chemicals that produce local increased cell turnover.

Genotoxicity Issuses

• Site of first contact carcinogens:– Generally require no metabolism to be carcinogenic;– Generally will produce tumors at other sites if the route

of administration is different tumor location is the site of contact;

– Generally only produce forestomach tumors in gavage/dietary studies because of limited/no systemic bioavailability;

– Typically alkylating agents;– Typically genotoxicants in vitro and in vivo;

– Forestomach tumours are potentially human relevant but only at the site of first contact in humans (e.g. dermal exposures)

Genotoxicity Issuses• Classical pro-carcinogen DNA reactive chemicals;

– Generally pro-carcinogens;– Often produce tumours at more than one anatomical site

following oral dosing (at least one systemic site + forestomach);

– Often other routes of administration also result in tumors;

– Generally systemically bioavailable;

– Human relevance of forestomach tumors depends on: (a) was there evidence of gastric irritation; (b) were the doses excessive (> MTD); (c) were the effects only seen with gavage dosing/diet studies and not with drinking water studies?

• Observation of tumours under different circumstances lends support to the significance of the findings for animal carcinogenicity. Significance is generally increased by the observation of more of the following factors:

• Uncommon tumour types; • Tumours at multiple sites; • Tumours by more than one route of administration; • Tumours in multiple species, strains, or both sexes; • Progression of lesions from preneoplastic to benign to malignant;

• Reduced latency of neoplastic lesions; • Metastases (malignancy, severity of histopath); • Unusual magnitude of tumour response; • Proportion of malignant tumours; • Dose-related increases;• Tumor promulgation following the cessation of exposure.

Benzo(a)pyrene (IARC 1)

Parameter

Genotoxicity in vivo that is relevant to humans +

Forestomach cancers following oral dosing +

Not observed in drinking water studies, only observed with gavage/diet studies -

Only observed at doses that irritate the forestomach (> MTD) -

Uncommon tumour types; +

Tumours at multiple sites; +

Tumours by more than one route of administration; +

Tumours in multiple species, strains, or both sexes; +

Progression of lesions from preneoplastic to benign to malignant; +

Reduced latency of neoplastic lesions; +

Metastases (malignancy, severity of histopath); +

Unusual magnitude of tumour response; +

Proportion of malignant tumours; +

Dose-related increases; +

Tumour promulgation following the cessation of exposure. +

Ethyl Acrylate

• Oral gavage: dose related increases in the incidence of squamous-cell papillomas and carcinomas of the forestomach were observed in rats and mice. Exposure caused gastric irritancy;

• Ethyl acrylate was tested by inhalation in the same strains of mice and rats; no treatment-related neoplastic lesions were observed;

• No treatment-related tumour was observed following skin application of ethyl acrylate for lifespan to male mice.

Ethyl Acrylate

Ethyl acrylate (IARC 2B)Parameter

Genotoxicity in vivo that is relevant to humans -


Not observed in drinking water studies, only observed with gavage/diet studies ?

Only observed at doses that irritate the forestomach (> MTD) +

Uncommon tumour types; -

Tumours at multiple sites; -

Tumours by more than one route of administration; -

Tumours in multiple species, strains, or both sexes; +

Progression of lesions from preneoplastic to benign to malignant; +

Reduced latency of neoplastic lesions; +

Metastases (malignancy, severity of histopath); -

Unusual magnitude of tumour response; -

Proportion of malignant tumours; -

Dose-related increases; -

Tumour promulgation following the cessation of exposure. +

Mercuric chloride (IARC 3)

Parameter

Genotoxicity in vivo that is relevant to humans -


Not observed in drinking water studies, only observed with gavage/diet studies

?

Only observed at doses that irritate the forestomach (> MTD) +

Uncommon tumour types; -

Tumours at multiple sites; -

Tumours by more than one route of administration; (thyroid follicular cell adenomas)

Tumours in multiple species, strains, or both sexes; -

Progression of lesions from preneoplastic to benign to malignant; -

Reduced latency of neoplastic lesions; -

Metastases (malignancy, severity of histopath); -

Unusual magnitude of tumour response; -

Proportion of malignant tumours; -

Dose-related increases; -

Tumour promulgation following the cessation of exposure. -

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Section 7:

Case Studies

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