1. TOXC 207 / PHCO 207 / ENVR 231Advanced Toxicology Biochemistry of Liver Injury Christopher Black, Ph.D.
Edward L. LeCluyse, Ph.D.
2. Effect of Toxic Chemicals on the Liver The liver is the most common site of damage in laboratory animals administered drugs and other chemicals.
There are many reasons including the fact that the liver is the first major organ to be exposed to ingested chemicals due to its portal blood supply.
Although chemicals are delivered to the liver to be metabolized and excreted, this can frequently lead to activation and liver injury.
Study of the liver has been and continues to be important in understanding fundamental molecular mechanisms of toxicity as well as in assessment of risks to humans.
3. Zonation of Liver Microstructure
4. Zonal Expression of P450’s
5. Chemical-induced Hepatotoxicity Hepatotoxic response depends on concentration of toxicant delivered to hepatocytes in the liver acinus
Hepatotoxicity a function of:
Blood concentration of (pro)toxicant
Blood flow in
Biotransformation (to more or less toxic species)
Blood flow out
Most hepatotoxicants produce characteristic patterns of cytolethality across the acinus
6. Types of Liver Injury or Responses Cell Death (necrosis, apoptosis)
Cholestasis (disrupted transport function)
Modulation of CYP activities (inhibition, induction)
7. Most Hepatotoxic Chemicals Cause Necrosis Result of loss of cellular volume homeostasis
Affects tracts of contiguous cells
Plasma membrane blebs
Increased plasma membrane permeability
Vesicular endoplasmic reticulum
Inflammation usually present
8. Necrosis Damage occurs in different parts of the liver lobule depending on oxygen tension or levels of particular drug metabolizing enzymes.
Allyl alcohol causes periportal necrosis because the enzymes metabolizing it are located there.
CH2=CHCH2OH CH2 =CHCHO
Carbon tetrachloride causes centrilobular necrosis - endothelial and Kupffer cells adjacent to hepatocytes may be normal - with diethylnitrosamine, endothelial cells are also killed. Due to activation by higher concentrations of cytochrome P450 in zone 3.
10. Chemical Exposure Can Also Lead to Apoptosis Defined primarily by morphological criteria:
Condensation of chromatin
Gene expression, protein synthesis
Ca++-dependent endonuclease activation
Cleavage to oligonucleosomes
Cytoplasmic organelle condensation
Death-receptor (TNF-R1, Fas) or mitochondrial pathways
Unlike necrotic cells, apoptotic cells show no evidence of increased plasma membrane permeability
11. Chemical-induced Hepatocyte Apoptosis
12. Apoptosis Mechanism
13. Fate of Injured Cells
14. LIPIDOSIS Many chemicals cause a fatty liver. Sometimes associated with necrosis but often not.
Not really understood but essentially is due to an imbalance between uptake of fatty acids and their secretion as VLDL.
Carbon tetrachloride can cause lipidosis by interfering in apolipoprotein synthesis as well as oxidation of fatty acids.
Other chemicals can cause lipidosis by interfering with export via the Golgi apparatus.
Ethanol can induce increased production of fatty acids.
15. Consequences of Toxic Mechanisms Disruption of intracellular calcium
Disruption of actin filaments
Generation of high-energy reactions
Covalent binding and adduct formation
Adduct-induced immune response
Cytolytic T cells and cytokines
Activation of apoptotic pathways
Programmed cell death with loss of nuclear chromatin
Disruption of mitochondrial function
Decreased ATP production
Increased lactate and reactive oxygen/nitrogen species (ROS, RNS)
Peroxidation of Membrane Lipids
Blebbing of plasma membrane
16. Mechanisms of Chemical-induced Toxicity Direct effects
Toxicants can have direct surfactant effects upon plasma membranes
Chlorpromazine and phenothiozines, erythromycin salts, chenodeoxycholate
Effects on the cytoskeleton, resulting in plasma membrane permeability changes
Effects upon mitochondrial membranes and enzymes
Cadmium, butylated hydroxyanisole, butylated hydroxytoluene, inhibitors and uncouplers of electron transport
17. Mechanisms of Chemical-induced Toxicity Alteration in the intracellular prooxidant-antioxidant ratio
Redox cycling of toxicant (e.g., quinone) produces oxygen radicals, depletes GSH
Hydroperoxides and metal ions (Fe, Cu) can produce oxidative stress and deplete GSH
Lipid peroxidation, protein sulfhydryl oxidation, disruption of Ca++ homeostasis
18. Redox Cycling and Formation of Oxygen Radicals
19. Critical Role of Glutathione Glutathione is the major cellular nucleophile, detoxication pathway for most electrophilic chemicals
Glutathione depletion generally makes cells more susceptible to electrophilic cellular toxicants, ‘threshold’ effect
Glutathione depletion induced by alkylating agents , oxidative stress, substrates, biosynthetically with buthionine sulfoximine
Glutathione can be increased by precursors, such as N-acetylcysteine, which is used as an antidote for toxicity
20. Mechanisms of Chemical-induced Toxicity Disruption of Calcium Homeostasis
Calcium regulates a wide variety of physiological processes
Ca++ accumulation in necrotic tissue, association with ischemic and chemical toxicity
Ca++ homeostasis in the cell very precisely regulated
Impairment of homeostasis can lead to Ca++ influx, release, or extrusion
21. Chemical Disruption of Ca++ Homeostasis Release from mitochondria
Uncouplers, quinones, hydroperoxides, MPTP, Fe+2, Cd+2
Release from endoplasmic reticulum
CCl4, bromobenzene, quinones hydroperoxides, aldehydes
Influx through plasma membrane
CCl4, CHCl3, dimethylnitrosamine, acetaminophen, TCDD
Inhibition of efflux from the cell
Cystamine, quinones, hydroperoxides, diquat, MPTP, vanadate
22. Consequences of Disruption of Ca++ Homeostasis Alterations in the cytoskeleton
Plasma membrane blebbing
Ca++ regulation of polymerization
Ca++-activated protease activity
Alterations in plasma membrane channels
Activation of phospholipases
Ca++- and calmodulin-dependent
Increased membrane permeability
Stimulation of arachidonate metabolism
23. Consequences of Disruption of Ca++ Homeostasis Activation of proteases
Calpain: Ca++-activated, non-lysosomal
Degradation of cytoskeletal and membrane proteins
Activation of endonucleases
DNA fragmentation, cell death
Acetaminophen, SDS, uncouplers
Possible mechanism of mutation induction by cytotoxic agents
24. Mechanisms of Chemical-induced Toxicity Reactive Metabolite Formation
Many compounds are metabolically activated to chemically reactive toxic species
Aflatoxin, carbon tetrachloride, acetaminophen, bromobenzene, nitrosamines, pyrrolizidine alkaloids
Chemically reactive metabolites (electrophiles) can covalently bind to crucial cellular macromolecules (nucleophiles)
Glutathione (GSH) is the prevalent cellular nucleophile, which acts as a protective agent
25. Covalent Binding Theory of Chemical Toxicity Metabolism of chemical to reactive metabolite
Covalent binding of reactive metabolite to critical cellular nucleophiles (protein SH, NH, OH groups)
Inactivation of critical cell function (e.g., ion homeostasis)
26. Immune-mediated Hepatotoxicity
27. Cytochromes P450 Prevalent heme-containing proteins of liver
Localized in the smooth endoplasmic reticulum
Many different forms with overlapping substrate specificity
Biosynthesis induced by treatment with a variety of xenobiotics
Induction can reduce or exacerbate hepatotoxicity
29. Biotransformation of Toxicants: Phase II Reactions ‘Synthetic’ reactions, conjugation with hydrophilic groups
Glucuronic acid, sulfate, glutathione, amino acids
Generally considered detoxication, water-soluble product
Can be metabolically activated to an unstable reactive product
30. Acetaminophen Metabolism and Toxicity
31. Acetaminophen Protein Adducts
33. Induction of Biotransformation Reactions Two major categories of CYP inducers
Phenobarbital is prototype of one group - enhances metabolism of wide variety of substrates by causing proliferation of SER and CYP in liver cells.
Polycylic aromatic hydrocarbons are second type of inducer (ex: benzo[a]pyrene).
Induction appears to be an environmental adaptive response to chemical insult
Receptors (AhR, PXR, CAR, PPAR) are regulators of genes involved in hepatic biotransformation reactions
34. Nuclear Receptors Involved in P450 Enzyme Induction
35. Consequences of Cytochrome P450 Enzyme Induction Increased toxic effect
Acetaminophen Alcohol, 3-MC
Bromobenzene, CCl4 Phenobarbital
Cyclophosphamide Macrolides, pesticides
Increased tumor formation
Altered disposition of endogenous substrates
Altered cell function
proliferation of peroxisomes and SER
increased liver weight
PCDDs, azobenzenes, biphenyls (PCBs), naphthalene
36. CYP 1A1 biotransformation PAHs from incomplete combustion undergo oxygenation to generate arene oxides
37. DNA adduct formation Reactive electrophiles bind covalently to DNA
39. Efflux pumps in hepatocytes Transporters and Xenobiotic Elimination
40. Ultrastructure of Bile Canaliculi in Hepatocytes
41. Potential Mechanisms for Cholestasis
42. Chemo-sensitization via Transporter Inhibition
44. Other Agents Causing Cholestasis in Animals Lithocholic acid – action can be reversed by cholic acid suggesting a competition for transport proteins
Ouabain – blocks Na+/K+ pump
Phalloidin and Cytochalasin B – Both affect actin microfilaments - possibly disrupting the actin corset around the bile canaliculus
Cyclosporin A – Causes symptoms of jaundice with no changes in the liver. Probably affects bile acid metabolism
45. Summary Biochemical mechanisms of hepatoxicity are complex
Some ‘classic’ cytotoxicity mechanisms and pathways
Some unique mechanisms and pathways
The observance of hepatoxicity is often a fine balance between multiple factors
46. Suggested Reading Jaeschke H, Gores GJ, Cederbaum AI, Hinson JA, Pessayre D, Lemasters JJ. Mechanisms of hepatotoxicity. Toxicol Sci. 65(2):166-76, 2002.
Klaassen CD, ed., Casarett and Doull’s Toxicology. The Basic Science of Poisons. 6th edition , McGraw Hill, New York, 2001.
Kim JS, He L, Qian T, Lemasters JJ. Role of the mitochondrial permeability transition in apoptotic and necrotic death after ischemia/reperfusion injury to hepatocytes. Curr Mol Med. 3(6):527-35, 2003.
Puga A, Xia Y and Elferink C. Role of AhR in cell cycle regulation. Chem-Biol Interact 141:117-30, 2002.
Hestermann EV, Stegeman JJ and Hahn ME. Relative contributions of affinity and intrinsic efficacy to AhR ligand potency. Toxicol App Pharmacol 168: 160-72, 2000.