TOXC 707/PHCO 707/ENVR 731TOXC 707/PHCO 707/ENVR 731Advanced ToxicologyAdvanced Toxicology

Biochemistry of Liver InjuryBiochemistry of Liver Injury

Edward L. LeCluyse, Ph.D.ed.lecluyse@cellzdirect.com

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.