TOXC 707/PHCO 707/ENVR 731TOXC 707/PHCO 707/ENVR 731Advanced ToxicologyAdvanced Toxicology
Biochemistry of Liver InjuryBiochemistry of Liver Injury
Edward L. LeCluyse, [email protected]
919-545-9959x306
Effect of Toxic Chemicals on the Effect of Toxic Chemicals on the LiverLiver
• 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.
Lobule
Zonation of Liver Zonation of Liver MicrostructureMicrostructure
Acinus
Chemical-induced Chemical-induced HepatotoxicityHepatotoxicity
• 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
Types of Liver Injury or Types of Liver Injury or ResponsesResponses
• Cell Death (necrosis, apoptosis)
• Cholestasis (disrupted transport function)
• Steatosis, Phospholipidosis
• Oxidative stress
• Mitochondrial dysfunction
• Modulation of CYP activities (inhibition, induction)
• Fibrosis/Cirrhosis
• Hepatitis
Most Hepatotoxic Chemicals Most Hepatotoxic Chemicals Cause NecrosisCause Necrosis
• Result of loss of cellular volume homeostasis– Affects tracts of contiguous cells– Plasma membrane blebs– Increased plasma membrane
permeability– Organelle swelling– Vesicular endoplasmic reticulum– Inflammation usually present
NecrosisNecrosis
• 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.
Chemical Exposure Can Also Chemical Exposure Can Also Lead to ApoptosisLead to Apoptosis
• Defined primarily by morphological criteria:– Condensation of chromatin
• Gene expression, protein synthesis• Ca++-dependent endonuclease activation• Cleavage to oligonucleosomes
– Cytoplasmic organelle condensation– Phagocytosis– Inflammation absent
• Death-receptor (TNF-R1, Fas) or mitochondrial pathways
• Unlike necrotic cells, apoptotic cells show no evidence of increased plasma membrane permeability
Chemical-induced Chemical-induced Hepatocyte ApoptosisHepatocyte Apoptosis
BileCanaliculus
Toxicant(TRZ)
TRZ
Ligand-independent Fas aggregation
Caspacecascade
Apoptosis
Vesicle With Fas
Jaeschke et al., Toxicol. Sci., 65:166, 2002.
Apoptosis MechanismApoptosis Mechanism
J. Biol. Chem., published online May 18, dx.doi.org/10.1074/jbc.M510644200
Fate of Injured CellsFate of Injured Cells
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.
Consequences of Toxic MechanismsConsequences of Toxic Mechanisms
• Disruption of intracellular calcium– Cell lysis
• Disruption of actin filaments– Cholestasis
• 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
Mechanisms of Chemical-Mechanisms of Chemical-induced Toxicityinduced 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
• Phalloidin, taxol
– Effects upon mitochondrial membranes and enzymes
• Cadmium, butylated hydroxyanisole, butylated hydroxytoluene, inhibitors and uncouplers of electron transport
Mechanisms of Chemical-Mechanisms of Chemical-induced Toxicityinduced 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
Redox Cycling and Formation Redox Cycling and Formation of Oxygen Radicalsof Oxygen Radicals
Critical Role of GlutathioneCritical 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
Mechanisms of Chemical-Mechanisms of Chemical-induced Toxicityinduced 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
Chemical Disruption of CaChemical Disruption of Ca++++ HomeostasisHomeostasis
• 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
Consequences of Disruption Consequences of Disruption of Caof Ca++++ Homeostasis 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
Consequences of Disruption Consequences of Disruption of Caof Ca++++ Homeostasis 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
Mechanisms of Chemical-Mechanisms of Chemical-induced Toxicityinduced 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
Covalent Binding Theory of Covalent Binding Theory of Chemical ToxicityChemical 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)
• Cell death
Immune-mediated Immune-mediated HepatotoxicityHepatotoxicity
From: Treinen-Moslen, Toxic responses of the liver, Casarett & Doull’s Toxicology, 6th Ed., 2001.
Cytochromes P450Cytochromes 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
Biotransformation of Biotransformation of Toxicants: Phase II ReactionsToxicants: 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
Metabolic Activation by Metabolic Activation by P450P450
• Formation of toxic species– Dechlorination of chloroform to phosgene– Dehydrogenation and subsequent epoxidation
of urethane (CYP2E1)
• Formation of pharmacologically active species– Cyclophosphamide to electrophilic aziridinum
species (CYP3A4, CYP2B6)
Liver Structure and Liver Structure and FunctionFunction
• Metabolic heterogeneity is responsible for zonal injuries – of value to the pathologist
• Zone 3 necrosis: acetaminophen; pyrrolizidine alkaloids; mushroom poisoning (A. phalloides); hydrocarbons – halothane and CCl4
• Zone 1 necrosis: allyl alcohol; phosphorous
• Zone 2 toxicity: rare
Zonal Expression of P450’sZonal Expression of P450’s
PVPV
CVCV
Labeling with P450 Antibodies
Acetaminophen Metabolism and Acetaminophen Metabolism and ToxicityToxicity
~60% ~35%
CYP2E*CYP1A CYP3A
NAPQIN-acetyl-p-benzoquinone imine
*induced by ethanol, isoniazid, phenobarbital
Protein adducts,Oxidative stress,Toxicity
HN
COCH 3
OH HN
COCH 3
OSO 3H
HN
COCH 3
OO CO 2H
OH
OHHO
N
O
COCH 3
Acetaminophen Protein AdductsAcetaminophen Protein Adducts
CYP2ECYP1ACYP3A
HS-Protein
H2N-Protein
S.D. Nelson, Drug Metab. Rev. 27: 147-177 (1995)J.L. Holtzman, Drug Metab. Rev. 27: 277-297 (1995)
HN
COCH 3
OH
N
O
COCH 3 HN
COCH 3
OH
S Protein
HN
COCH 3
OH
NH Protein
O
COCH 3NSProtein
Induction of Biotransformation Induction of Biotransformation ReactionsReactions
• 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
Nuclear Receptors Involved Nuclear Receptors Involved in P450 Enzyme Inductionin P450 Enzyme Induction
CYP4ALipid metabolism
RXRPPAR
CYP2BXenobiotic, Steroid metabolism
RXRCARConstitutive AndrostaneReceptor
Peroxisome Proliferator-activated Receptor
CYP3AXenobiotic, Steroid metabolism
RXRPXRPregnane XReceptor
CYP1AXenobiotic metabolism
AhR ARNTAryl HydrocarbonReceptor
Consequences of Cytochrome Consequences of Cytochrome P450 Enzyme InductionP450 Enzyme Induction
Consequences of Cytochrome Consequences of Cytochrome P450 Enzyme InductionP450 Enzyme Induction
• Increased toxic effect– Acetaminophen Alcohol, 3-MC
– Bromobenzene, CCl4 Phenobarbital
• Increased bioactivation– Cyclophosphamide Macrolides, pesticides
• Increased tumor formation– Altered disposition of endogenous substrates
• Altered cell function– proliferation of peroxisomes and SER– increased liver weight
• Porphyria, chloracne
• PCDDs, azobenzenes, biphenyls (PCBs), naphthalene
CYP 1A1 biotransformationCYP 1A1 biotransformation
• PAHs from incomplete combustion undergo oxygenation to generate arene oxides
B[a]P
(Cavalieri & Rogan, 1993)
Peroxidases Oxidants CYP
1A1
B[a]P radical cation+
e-
OH
CYP1A1 and
epoxide hydrolase
HO
O
B[a]P diol epoxide
DNA adduct formationDNA adduct formation
• Reactive electrophiles bind covalently to DNA
B[a]P radical cation
+
Guanine
HN
N
N
NH
O
H3C
.. HN
N
N
NH
O
H3C
B[a]P-6-N7Gua
(Cavalieri & Rogan, 1993)
HepatocyteBile
Canaliculus
Na+
Na+
K+
TJ
Mrp3
Ntcp
Oatp
Oct
Oat
Mrp1
Mrp5 Mrp6
Sinusoidal and Canalicular Sinusoidal and Canalicular Membrane Transport Proteins of Membrane Transport Proteins of
the Hepatocytethe Hepatocyte
Hepatocyte
Mrp2
Bsep
Mdr1
BCRP
Efflux pumps in hepatocytes Efflux pumps in hepatocytes
Transporters and Xenobiotic Transporters and Xenobiotic EliminationElimination
Ultrastructure of Bile Canaliculi Ultrastructure of Bile Canaliculi in Hepatocytesin Hepatocytes
Tight & AdherenceJunctions
X
Potential Mechanisms of CholestasisPotential Mechanisms of Cholestasis
From: Treinen-Moslen, Toxic responses of the liver, Casarett & Doull’s Toxicology, 6th Ed., 2001.
Chemo-sensitization via Chemo-sensitization via Transporter InhibitionTransporter Inhibition
From Vega, R. L., Stanford University Hopkins Marine Station, Pacific Grove, CA;2004 EPA Graduate Fellowship Conference.
Hepatobiliary Transporters and Hepatobiliary Transporters and ToxicitiesToxicities Transporters involved in hepatic CL may determine
systemic exposure and bioavailability e.g. statins (OATP transporters implicated)
Hepatic accumulation may result in hepatotoxicity e.g. methotrexate (MRP2 implicated?)
Inhibition of transporter activity may result in cholestasis e.g. bosentan (BSEP implicated)
Inhibiting transporter activity may result in hyperbilirubenemia e.g. indinavir (OATP1B1 implicated)
Inhibition of transporter activity may result in toxic DDI e.g. gemfibrozil and cerivastatin (OATP1B1
implicated)
Concentrative biliary excretion may cause GI toxicity e.g. irinotecan (MDR1/MRP2 implicated)
Other Agents Causing Other Agents Causing Cholestasis in AnimalsCholestasis 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
SummarySummary • 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– Toxicokinetic– Environmental– Physiological
Suggested ReadingSuggested 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.
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