PXR assay (PowerPoint)

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					TOXC 207 / PHCO 207 / ENVR 231
     Advanced Toxicology

 Biochemistry of Liver Injury
        Christopher Black, Ph.D.
                  for
       Edward L. LeCluyse, Ph.D.
          edl@cellzdirect.com
           919-545-9959x306
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.
Zonation of Liver Microstructure



              Acinus




     Lobule
Zonal Expression of P450’s

                          Labeling
                         with P450
                         Antibodies
  PV
                  CV
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
 Types of Liver Injury or Responses
• 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
         Cause 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
                      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.
   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
  – 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 Hepatocyte
                  Apoptosis
                              Toxicant               Ligand-independent
                               (TRZ)                   Fas aggregation


          Bile
       Canaliculus
                                                            Caspase
                                        TRZ    Vesicle     cascade
                                                With Fas



                                                Apoptosis

Jaeschke et al., Toxicol. Sci., 65:166, 2002.
                          Apoptosis Mechanism




J. Biol. Chem., published online May 18, dx.doi.org/10.1074/jbc.M510644200
Fate 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 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-
          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
      • Phalloidin, taxol
   – Effects upon mitochondrial membranes and
     enzymes
      • Cadmium, butylated hydroxyanisole, butylated
        hydroxytoluene, inhibitors and uncouplers of electron
        transport
     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
Redox Cycling and Formation of
      Oxygen Radicals
    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
    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
    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
       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
       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
     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
    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)
• Cell death
       Immune-mediated Hepatotoxicity




From: Treinen-Moslen, Toxic responses of the liver, Casarett & Doull’s Toxicology, 6th Ed., 2001.
          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
 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
     Acetaminophen Metabolism and Toxicity
                                   COCH 3
                              HN



                    ~60%                        ~35%
     COCH 3                     OH
HN
                                                            COCH 3
                                   CYP2E*
                                                       HN
                                   CYP1A
                                   CYP3A
       O      CO 2 H
 O
                                       COCH 3
              OH                   N                    O
HO                                                          SO 3 H
       OH
                                           *induced by ethanol, isoniazid,
                                            phenobarbital
Protein adducts,                   O
                                 NAPQI
Oxidative stress,
                     N-acetyl-p-benzoquinone imine
Toxicity
       Acetaminophen Protein Adducts
     COCH 3                               COCH 3
HN                                    N


                 CYP2E                             HS-Protein
                 CYP1A
  OH             CYP3A                O
                                                     H2N-
                                                     Protein

              COCH 3         COCH 3                         COCH 3
Protein S N             HN                            HN



                               S Protein                     NH Protein
        O                OH                             OH


       S.D. Nelson, Drug Metab. Rev. 27: 147-177 (1995)
       J.L. Holtzman, Drug Metab. Rev. 27: 277-297 (1995)
  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
          Nuclear Receptors Involved in
            P450 Enzyme Induction
   Aryl Hydrocarbon        AhR ARNT               CYP1A
       Receptor                            Xenobiotic metabolism



Constitutive Androstane    CAR RXR
                                                   CYP2B
       Receptor                        Xenobiotic, Steroid metabolism



      Pregnane X           PXR   RXR               CYP3A
       Receptor                        Xenobiotic, Steroid metabolism



Peroxisome Proliferator-                          CYP4A
                           PPARa RXR
  activated Receptor                       Fatty acid metabolism
 Consequences of Cytochrome
   P450 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 biotransformation


•   PAHs from incomplete combustion undergo oxygenation
    to generate arene oxides




    B[a]P                                   O
                          CYP1A1 and
                     epoxide hydrolase

            Peroxidases                HO
                                                     B[a]P diol epoxide
             Oxidants
                                                OH
             CYP 1A1

             e-

                                  B[a]P radical cation
                          +

                                       (Cavalieri & Rogan, 1993)
                    DNA adduct formation


• Reactive electrophiles bind covalently to DNA




B[a]P radical
                                 B[a]P-6-N7Gua
cation

                +

                                          O
         O
                    ..
                    N                HN
                                                 N

    HN
                                          N      N
                                   H3C
         N    N                                  H
  H3C
              H
         Guanine

                               (Cavalieri & Rogan, 1993)
Sinusoidal and Canalicular Membrane
Transport Proteins of the Hepatocyte
         Na+



   Na+           K+          Mrp1
                      Mrp3
Ntcp                                          Mrp2

                                                     Bsep
Oatp
                                       Bile
               Hepatocyte                             Hepatocyte
                                    Canaliculus
Oct

                                                  Mdr1
 Oat                                         BCRP
                      Mrp5 Mrp6         TJ
    Transporters and Xenobiotic
           Elimination
Efflux pumps in hepatocytes
Ultrastructure of Bile Canaliculi
         in Hepatocytes


         X




                 Tight & Adherence
                     Junctions
                     Potential Mechanisms for
                            Cholestasis




From: Treinen-Moslen, Toxic responses of the liver, Casarett & Doull’s Toxicology, 6th Ed., 2001.
         Chemo-sensitization via Transporter
                    Inhibition




From Vega, R. L., Stanford University Hopkins Marine Station, Pacific Grove, CA;
     2004 EPA Graduate Fellowship Conference.
Hepatobiliary Transporters and Toxicities
 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
        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
                 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
  – Toxicokinetic
  – Environmental
  – Physiological
             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.

				
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