TOXC 207 / PHCO 207 / ENVR 231 Advanced Toxicology Biochemistry of Liver Injury Christopher Black, Ph.D. for Edward L. LeCluyse, Ph.D. firstname.lastname@example.org 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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