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Acs Short Courses American Chemical Society Chemical Mechanisms in Toxicology

Date: Jul 1995 (est.)
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Bond, J.
Brusick, D.J.
Dietert, R.R.
Grippo, J.F.
Hodgson, E.
Klaassen, C.D.
Korzekwa, K.R.
Mcclellan, R.O.
Moslen, M.T.
Slikker, W., J.R.
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Bacon
Bentley
Britton
Brown
Brusick, D.J.
Christen
Cotran
Curnutte
Dipple, A.
Doull
Duchesne
Ecobichon, D.J.
Fee
Frank
Fridovich, I.
Gasarett
Gelvan
Gilman
Goodman
Gorelick
Griffith
Grippo, J.F.
Gudas
Hafez
Harris
Hodgson, E.
Hooper
Hoppeseyler
Keshan
Klaassen, C.D.
Klein
Kukovetz
Kushi
Levi, P.E.
Longmire
Mccord, J.M.
Meister
Menten
Michaelis
Michelson, A.
Morrow
Moslen, M.T.
Oberley
Parkinsons
Politzer, P.
Pratt
Pryor
Robbins
Roberts, L.
Seekamp
Setlow
Squatrito
Stone
Taylor
Tokumaru
Turner
Valentine
Wilms
Young
Hanulcoglu
Osborne
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Am J Clin Nutr
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ACS Short Courses American Chemical Society CHEMICAL MECHANISMS . IN TOXICOLOGY co ~ Dr. James Bond ~ Course Director ~ ~ ~
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Moslen - I Page 1 0 Free Radicals Mary Treinen Moslen, Ph.D. William C. Professor of Environmental Toxicology Director, Toxicology Training Program University of Texas Medical Branch Galveston, Texas 0 Mary Treinen Moslen was born in Iowa, received a BA in chemistry from Vassar College, a MS in scientific communication from Boston University, and a PhD from UTMB. Her research interests are cell injury and toxicant effects on bile formation. She is a member of the Society of Toxicology and the Society of Investigative Pathology. Her service on national committees included membership on the NIH Toxicology Study Section. She has served on the editorial boards of several journals and as an associate editor of Toxicology and Applied Pharmacology. 00 w Lv ~ CO C:) CD n N)
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06 Lecturer JAMES BOND MARY TREINEN MOSLEN JOSEPH F. CRIPPO JOSEPH F. GRIPPO DAVID J. BRUSICK ERNEST HODGSON KENNETH KORZEKWA KENNETH KORZEKWA CURTIS D. KLAASSEN CURTIS D. KLAASSEN RODNEY R. DIETERT WILLIAM SLIKKER, JR. WILLIAM SLIKKER, JR. MARY TREINEN MOSLEN ROGER 0. MCCLELLAN TABLE OF CONTENTS Topic INTRODUCTION FREE RADICALS RECEPTORS IN TOXICOLOGIC MECHANISMS RETINOIDS AND TERATOGENESIS: MOLECULAR APPROACHES AND TECHNIQUES CENOTOXICOLDCIC MECHANISMS PESTICIDES: METABOLISM AND MECHANISMS OF TOXICITY METABOLIC FORMATION OF CHEMICALLY REACTIVE SPECIES THE CYTOCHRONE P450 ENZYMES AND CHEMICAL CARCINOCENESIS MECHANISMS OF LIVER TOXICITY MECHANISMS OF KIDNEY TOXICITY IMMUNOTOXICOLOGIC MECHANISMS MECHANISMS OF NEUROTOXICITY POSTULATED MECHANISMS OF NEUROTOXICITY PROTECTION AGAINST FREE RADICAL MEDIATED TISSUE INJURY INHALATION TOXICOLOGY a
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0 Production of ROS by Redox-Recycling Semiquinone Free Radical O- i Production of ROS by Activated Neutrophils Elastase Collagenase Moslen - I Page 5
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Free Radicals Formation of Reactive Oxygen Species (ROS) A. B. Pathways Macrom4lecular Targets 1. Lipids 2. Proteins 3. DNA II. Consequences of ROS III. Oxidized Metabolites of Nitrogen A. B. Formation Toxicity IV. Measurement of ROS and Products Moslen - I Page 2 0 0 !
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Moslen - I Page 3 ! Excitation and Reduction of Molecular Oxygen Singlet oxygen 4-d:0't 0:0: s H02 +H //- H+ • 02~02 e 4'Q Q't '9:0: Oxygen Superoxide anion -H2O2-e-0H•-eH2O H:O:O~H •O:H H:O:H Hydrogen Hydroxyl Woter peroxide radicai OH° )100~ 2GSH\ Catalase Glutathione Giutathione peroxidase reductase v GSSG...W H20
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Production of ROS by Iscitemia-Reperfusion . ~ ~ ~ x ~ mitochondrial injury W "ieaky" electron transport 02 ATP ~ AMP Adenosine T Inosine Xanthine Dehydrogenase Xanthine Oxidase ~ E----~,,_ /~ Hypoxan#h'rne REPERFUSION 7 REPERFUSION PRIas#en Page 6 Urate -f- i !
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Moslen - I Page 7 0 Formation of Nitric Oxide by Nitric Oxide Synthetase (NOS) L-Arginine i L-Arginine RS -.NO• _ I\~0z• NADPH NADP L-NAME 02 or L-NME 0.0 0.5 1.0 VASCULAR PERMEABILITY INDEX Figure from Seekamp et al, 1993 •OONO i Intracellular Targets Effects on ischemia-Reperfusion Injury in Rats treated with modulators of Arginine L-Synthetase L- Citrulline ~ RS-NO~ ~ NOS L-NMA LUNG L-NAME a L-arqlnlne • Poelttve Contro/ Target Cell i
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~ Wrecking Crew of Reactive Oxygen Species (ROS) Reduction Potentials for Oxygen Species 0 O? + e ~ 02 • EEO'= -0.33 OZ-- +e+2H'~ H2Oz F~'=+0.87 H2OZ+e+H`~•OH+HzO EQ'=+0.38 •Ol-I + e + Hk ~ H20 EQ'= +2.33 czo w w 0c, All reactions at pH 7.0 0 0 ~ From Fee and Valentine, in Superoxide and Superoxide Dismutases (A. Michetson, J.M. McCord, and [. Fridovich, eds.), pp. 19-60, Academic Press, NewYork, 1977. Moslen - I Page 12
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Moslen - I Page 11 40 Macromolecular Targets of ROS: DNA N ~-OH N R R 0 R R Oxidation of guanosine to 8-hydroxyguanosine by singlet oxygen [IOZ] or •OH. 5-Hydroxycytosine H 5,8-Dihydroxyuracil (5R-andSS-) W W CYS Representative reactive oxygen species-modified DNA bases. ~ Thymine glycol HO,, C ~ ) H1 (cis- and trans N 11 OH H 2 HO"-~N~ ~N~ $•5'-Gyclo-c-aeoxyguanosme ®© 2-Hydroxyadenine
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Oxidized Metabolites of Nitrogen: Nomenclature Symbol Name Comments NO' nitric oxide free radical N0Z- nitrogen dioxide free radical, nitrosating agent N20 nitrous oxide anesthetic (laughing gas) N204 dinitrogen tetroxide nitrosating agent (dimeric N02• ) NOZ - nitrite produces NO' at acidic pH N03 - nitrate stable ion Oxidized Metabolites of Nitrogen: Reactions H2 0 2N0• + OZ--3m- 2NO2' HZO 2NO2• -30 NZ04 ~ N204 + N03 - RZ-NH + NOZ' -3io R2-N-N=O + 02 ' + NO' _~ ONOO' Moslerv - I Page 8 • 0 N02• + HO'-.*-ONOOH L
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Moslen - I Page 4 When and How ARE Reactive Oxygen S ecies Formed? Exposure to: R'HEN? Sunlight, Radiotherapy Drugs, Pollutants, Cigarette Smoke Inflammation, IschemialReperfusion Deficiency of Antioxidants, Excess of Iron 7 Processes of: r:rrts L,l.f,w2 rm A Mitochondrial Electron Transport Cytochrome P450, Redox Recyling Reactions NADPH Oxidase, Myeloperoxidase Xantlrine Qxidase Nitric Oxide Synthetase CR ) 16 0 Superoxide Production by the Mitochondrial Electron Transport Chain NADH dehydrapenasa Ubictuinone cyt t. cyt eyt a a cyt a3
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Macromolecular Targets of RJS: Proteins Vulnerabie sites include cysteine and histidine v Cr,' Examples XX ~ ~ t~, Ca-ATPase --` " Metal Binding Proteins Superoxide Dismutase Catalase Glucose-6-Phosphatase Many Other Enzymes Peroxynitrate oxidation of methionine to a sulfoxide. R2$~+ d j2b--NQ-~ H RzS-tJFi + NOZ R2S--O + H` Peroxynitrite can alter the aromatic ring of an amigo acid by hydroxylation or nitration. N *p 0 s Pryoz and Squatrito, Am J Physio1268:L699, 1995
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Moslen - I Page 13 0 Effects of Peroxidation on a Polyunsaturated Fatty Acid? > LIPID PEROXIDATION REACTIONS NV°V_W"NQ MV~v-v'v'vr0 i PEPOIODATION Polyunsaturated fatty acid of a membrane phospholipic is converted to lipid radical by an "initiation" reaction with •OH. The lipid radical is converted to a lipid peroxy radica. (LOO•) by a"peroxidation" reaction with OZ. Note the shift of a double bond to form a conjugated diene. Lipid peroxy radicals are highly unstable species which will abstract a hydrogen from a nearby lipid species (LH) in a"propagation" reaction to form a lipid radical (L •) plus a lipid hydroperoxide (LOOH). Alternatively the unstable lipid peroxy radical will rearrange to form unstable endoperoxides that break to form aikoxy radicals (LO•), and will eventualiy ,. 1. 0 ar nV-v°vw-v'q o, decompose to shortened oxidized phosphohpids, smali ~~J reactive aidehydes (eg, malondialdehyde) , and reactive alkenals (eg, 4-hydroxynonenal). Also Fe can stimulate <A the decomposition of lipid hydroperoxides. 9~„Syd> M~ 4_1~
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Moslen - I Page 17 Detecting Products Formed by ROS - 2 9 Thiobarbituric acid reacts with aldehydes to form a readily measured pink product This assay, often called TBARS is typically used as an indices of MDA. H 2 . Parinaric Acids (cis and trans) are extensively unsaturated compounds which are susceptible to oxidation; this property was used to develop assays for lipid hydroperoxides. Can be difficult to use since must be stored in argon and under light protection. A Lucinogen emits chemiluminescence upon oxidation by superoxide. Luminol, another chemiluminescence probe, emits light when oxidized by superoxide or hydrogen peroxide. Aging has been associated with increased ROS release by neutrophils of both men and women using a luminol assay. O 12 v~ 10 • females ~ S ~ males 2 N03 -pb 6 Ct) 4 b W 2 . . W co 0 O C7 3 0 20 40 60 s0 100 Age of subjects r Lucinogen Kukovetz et al _ Free Rad Biol Med 22:433,1997
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Moslen - II Page 3 0 PRIMARY AND AUXILIARY PROTECTION SYSTEMS Primary Protection Systems Antioxidants Vitamin E (Tocopherol) (3-Carotene Glutathione (GSH) Vitamin C (Ascorbate) Antioxidant Enzymes Superoxide Dismutase Enzymes (DOD) Catalase Glutathione Peroxidase Glutathione Transferase Aldehyde Dehydrogenase Transition MetaI Binders ~„ Transferrin ~ Ferritin Ceruloplasmin Albumin Metallothionein Auxiliary Protection Systems Antioxidant Regenerators MISC GSSG Reductase Phospholipase Glucose-6-P-Dehydrogenase GSSG and GS-Conjugate Exporters Membrane Radical Reductase .1 Modified from Moslen, 1992
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Moslen - I Page 9 Macromolecular Targets of ROS: Lipids 9 Free radical-initiated peroxidation of an unsaturated fatty acid. H H _• + ~__ \ .o-O~i\' ~~ ~E;~'' H H + ~`~_.~ Ho-o ~'_z0niJi= Reactive oxygen species-mediated conversion of cholesterol to 7-peroxide radical, 7-peroxide, 7-keto and 7-hydroxy products. m HO IIHO ` 00• ~ . 0 HO
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lv14B.;t6-gI - dd 3'age ;, How Are Reactive Oxygen Species Detoxified ? 02 P450 oxidase NADPH oxidase Respiratory chain enzymes Cytosolic enzymes Oxidase OH& ~ 2GSH \ Glutathione Glutathione peroxidase reductase I ~GSSG~ H20 ! ! The reactive oxygen species •OZ and HZOZ are formed by reactions in the endopiasmic reticulum (ER), mitochor)dria (MIT), and peroxisomes of cells. The very reactive radical .OH Is formed by electron transfer to HZOZ with [t=e4~ - frequently acting as the electron donor. The Fe•++ formed is cycled back to Fe" a' by •OZ . c.y w 00 •OZ is detoxified by the enzyme superoxide dismutase (SOD) many orders of o magnitude faster than the spontaneous disrriutation reaction. CD HZOZ is detoxified either by catalase or glutathione peroxidase with 4hr oxidation of GSH to GSSG. The GSSG formed is regenerated back to GSi-0 !~y the enzyme glutathione reductase •OH is extremely reactive and not readily detoxified. (Diagram from Robbins et al, 1989) C-) 0
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Moslen- .I1 Page 2 Protection Against Free Radical Damage 0 I. IL Types of Protection Against Free Radicals A. Detoxification of Reactive Oxygen Species (ROS) B. Control of ROS Formation by Phagocytes Control of Lipid Peroxidation A. Detoxification of the Products of Lipid Peroxidation B. Consequences of Inadequate Control L. Keshan's Disease 2. Deficiency of Glucose 6-P Dehydrogenase III. IV. Distribution of Protective Systems Within Cells and Tissues Adaptive and Therapeutic Protection 0 V. VI. Systemic Control of Fe Regulation of Fe Homeostasis Consequences of Excess Fe in Hemochromatosis Smoking, Antioxidants, and Cardiovascular Disease A. Insight into Black Box B. Therapeutic Needs
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What Processes Use and Restore Gtutathione ? YN7p4. GLU, CYS, GLY 1 SYNTHESIS UTILIZATION REGENERATION 0 • What Supplies the "Fuel" to Regenerate GSH ? Regeneration of GSH by G6PD GSSG glutathione reductase / NADPH, c~,., w ~ glucose-fi-P C.Ij dehydrogenose ~° GSH ` NADP MosYgn - II Page 12 'LIJCOSE
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Moslen - I Page 16 Detecting Products Formed by ROS - l Basic approaches for detection of free radical reaction products: • direct - absorption spectra, HPLC, GC-MS • conjugated - colored or fluorescent species • amplified - luminescent • antibody - ELISA or immunohistochemistry Lipid+ CI- CCl4 ~ •CCls j ~~ CHC13 s CFsCHC1Br T- CF3CHCl CF3CH2Cl Br Products formed by the bioactivation of carbon tetrachloride and halothane to free radicals which abstract hydrogen atoms from lipids. I* ! 10
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Wrecking Crew of Reactive Oxygen Species (ROS) Moslen-II Page 5 Superoxide anion radical, .Oz , can be considered as the master radical because it gives rise to other radicals. Hydroxy radical, *OH, is considered the most reactive and 'dangerous ROS species because contact with •OH damages almost any biological molecule. 9 Hydrogen peroxide, H202, results from radicals and readily generates other radicals. H1OZ can cross membranes and begin the oxidative chain reactions that damage membranes. Lipid peroxy radical, LOO., represents the radical formed in lipid peroxidation reactions. ~ LOOa has a surprisingly long half life of seven Cj seconds. Cartoon slightly modified from Hooper (1989).
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Moslen - I Page 15 • Consequences of ROS . •OZ , •OH, R• ~ i Olridized Membrane - Dysfunction '`- 3111- Cytotoxicity ~ .. Protein Dysfunction ~ Atherosclerosis ,~ Cataracts / Other Health Impaired Problems DNA Repair • ~ Oxidized Lipid Proteins Perolddation ~l Aldeh desi~ i Alkenals f / Teratology Mutation P-~ f Neoplasia
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Moslen - II Page 13 • Why Export GSSG and GS-Conjugates ? * GSSG can interact with protein thiols (RSH) to form mixed disulfides (R-S-SG). * GS-conjugates can inhibit both glutathione • transferase and GSSG reductase enzymes. GS-Conjugate . ~ GS-Conjugate Exporter How Are GSSG and GS-Conjugates Exported ? * ATP-dependent transporters move GSSG and GS-conjugates out of cells. * Hepatocytes preferentially transport GSSG and GS-conjugates into bile which is a direct route of hepatocyte excretion. trJ c,a w * Heart efficiently exports the GS-conjugate of ~ 4-hydroxynonenal, a toxic product of lipid ~ oxidation.
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Moslen - H Page 23 0 What Is Hemochromatosis ? * Common genetic disease of middle age that may affect 1 in 220 whites of northern European ancestry. * Characterized by hepatic fibrosis or cirrhosis, dilated cardiomyopathy, skin pigmentation, diabetes, arthritis, and testicular atrophy. * Etiology is accumulation of Fe in liver and then other tissues with diminished GI excretion from sloughed enterocytes. * Genetic defects appear to be related to the enterocyte gene products that regulate enterocyte uptake and/or storage of excess circulating iron. * Prognosis is good with early diagnosis and treatment by maintenance phlebotomies every 3-4 months. How Does the Body Control Fe Levels ? ~ * Uptake of dietary Fe is regulated by the enterocytes of the of the intestinal mucosa. When body Fe levels are low, uptake of dietary iron is enhanced and less Fe is stored in the enterocytes. * Plasma Fe is largely tightly bound to transferrin. * Transferrin-bound Fe enters cells by receptor-mediated endocytosis. * Within cells, Fe is incorporated into the many Fe-dependent proteins. Excess Fe is stored within ferritin molecules. With further accumulation, the excess Fe is stored as hemosiderin within lysosomes. * Free Fe levels are normally kept low by the combination of extra- and intra- cellular binding proteins. * Systemic Fe levels can be lowered by processes regulated by enterocytes. Specifically, enterocytes can up regulate synthesis of both the serosal transferrin receptor and the Fe binding protein, ferritin, which would enhance systemic Fe uptake and storage. * Fe stored in enterocytes is "excreted' into the intestinal lumen when "aged" ~ enterocytes are extruded into the intestinal lumen. t,.i. 40 CO n Cn .E1~ V
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Moslen - II Page 9 LLPID PERQxIDnTt4N REACTIONS lvYYYVahQ ~ xxnanow • Rp W-W-v-V-AA~ r ~ ~.~E~,~ PHOSPHOlIVASE co U-i U4 co ~ CN
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Antioxidants and Heart Disease - 1 Deaths Attributed to Smoking Active "Smoker" Passive "Environmental Smoker" Cardiovascular Disease 201,000 37,000 Lung Cancer 112,000 3,800 Chronic Lung Disease 83,000 ? Other Cancers 31,000 ? Total Deaths 430,000 > 50,800 Estimates based on 1988 deaths of adults > 35 years old Taylor et al, Circulation 86: 699-702, 1992 i 0 PLAQUE INITIATION & PROMOTION Moslen - II Page 27
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INTESTINAL LUMEN BLOOD INTESTINAL lUk1EN Fil« When Fe Excess 0 Increased Uptake from Stood @ Increased Storage in Ferritin BLggg Mosten - L! Page 24 ! 0
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M€sslen _ @1 Page ;~ What Processes Control ROS Formation by Phagocvtes? B) Confinement Within a Phagolysosome . . .. . P MyN~Y~me Attachment 1~1'\ s . . ~ . 1arlrtaclon ana ~ . ' degranuiabon 0 ! Cartoon of sequential events in the attachment, engulfment and phagocytosis of a particle by a phagocyte. Bacterial particle attachment stimulates activation of the NADPH oxidase that is localized to the phagolysosomal membrane. In addition, granules fuse with the phagocytic vacuole and release their contents into the phagolysosome. Contents of these granules include potentially destructive lysozyme, collagenase, elastase, phospho(ipase and myeloperoxidase, but also lactoferrin which binds up free Fe. This confinement within a phagolysosome limits, but does not total prevent, the escape of reactive oxygen species and other granule contents to the extracellular during phagocytosis. One type of powerful phacocytic cell, the neutrophil, is considered a"kamikaze" because it kills itself by apoptosis within 48 hr after attacking phagocytic particles. (Figure from Cotran et a(, } 989} 0
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Antioxidants and Heart Disease - 6 Tocopherols: y versus a a is the more potent antioxidant in vitro a is the primary form in Vitamin E supplements a to y ratio in most tissues is 5 times ( ie, 5x more a) possibly due to greater incorporation of a into nascent VDLD a to y ratio increases to > 20 with Vitamin E supplements y is the principal form in the US diet y is displaced in LDL and other sites by high levels of a y is a more effective inhibitor'of HOONO-mediated oxidation of phospholipid ' y is a more potent inhibitor of NO2• -mediated DNA strand breaks y reacts differently with HOONO than a Y1' ddo=+ Hzo Moslen -,IT Papp 32 nxTQ 0 • Christen et al, PNAS 94: 3217, _';'97 0
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Moslen - II Page 25 . HEMOCHROMATOSIS I Lipid Peroxidation of Organelle Membranes Direct Effect of Collagen Synthesis and/or ..., Degradatio ~n Increased Lysosomal Decreases in ~ Alterations in Fragility Mitochondrial Oxidative Microsomal •'Metabolism Enzymes, Cytochromes 0 Decreased Fe Excretion Hepatic Parenchymal Fe Overload Cell Injury, Cell Death -o~ Fibrosis, Cirrhosis -4 l Hepatocellular Carcinoma Proposed pathophysiological mechanism of liver injury in chronic iron or copper overload. Note the progression from cell injury to fibrosis and then to neoplasia. Modified from Britton and Bacon, 1990. co cA ~ cl-A co ~ CD ~ ~ -7J93 Acs
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How Is Vitamin E Regenerated ? r ! Regeneration of membrane Vitamin E (E) from Vitamin E radical (E.) by cytosolic Vitamin C(C). The Vitamin C radical (C•) rearranges to the stable species dehydroascorbate (DHA). Moslen - II Page 11 Urinary Excretion of F=-laoproatances by Smokers Urinary F3-Isoprostanes pmol/mmol creatinine Treatment (N) Before Treatment After Treatment P Vitamin E (7) 172 f 30 163 t 28 NS V itamin C (5) 195 f 41 137 t 34 < 0.05 Vitamin E+ C (4) 171 f 40 133 t 30 < 0.05 CO f ni Values are means ± SE . CKI Circulation 94:19-25, 1996. bd
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Moslen -11 Page 21 i ! 2501 2o 0 SOD Catalase +p(0.05 24 24 72 Glutathione Peroxidase * 24 72 Hours of Reexposure to 95% 02 3) Why are enzymes, as delicate molecules, such an important aspect of the adaptive response to ROS ? a) Antioxidant enzymes are inducible. b) As aptly stated by Harris (1992), "enzymes are designed specifically to execute reactions ~ with speed, specificity, and high affinity" which are "desirable properties of any How Do Organisms Adapt to ROS Exposure? 1) Protocols which produce adaptation to high oxygen. a) Exposure to gradually increasing oxygen concentrations b) Challenge with a high oxygen concentration and then a brief rest before sustained exposure to high oxygen concentrations. For example, 48 hrs of 95% oxygen challenge and then a 24 hour rest. c) Lungs of adapted animals, which are the target tissues for oxygen toxicity, show elevated activities of one or more antioxidant enzymes. 2) Patterns of enzyme induction reported by Frank et al (1989) in the lungs of. rats who were previously adapted to 95% oxygen by a 48 hr challenge and then a 24 hr rest protocol. 300r antioxidant". 83380045
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Moslen - II Page 29 0 . • Antioxidants and Heart Disease - 3 Cigarette smoke oxidizes LDL in vitro Oxidation alters the fate of LDL Key differences are: v uptake by scavenger receptors on macrophages within subendothelial spaces of arteries v macrophages laden with ox LDL become foam cells in plaques v ox LDL injure aortic muscle cells and cause plaque growth Cytotoxic species in LDL include oxysterol products of cholesterol lmmunohistochemistry with antibodies to ROS modified entities allows characterization of the sites and extent of ROS modification of macromolecules in vascular spaces. Antibodies raised against oxidized LDL react with atherosclerotic lesions but not against normal appearing arterial segments. e 0 0 Cytotoxicity of Oxidized LDL and its Oxysterol Constituents to Aortic Smooth Muscle Cells 90% N-LDL Ox-LDL 7-Keto Chol 7-OH Chol 100 ug/ml 100 ug/ml 5 ug/ml 2.5 ug/ml Arterioscler Thromb 14: 1177, 1994
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D INITIATION PEROXIDATION PROPAGATION DECOMPOSITION > UPID PEROXlDATlON REACTIONS Moslen - TLI Page 10 Polyunsaturated fatty acid of a membrane phospholipid is converted to lipid radical by an "init€ation" reaction Vrth • 4H. The lipid radical is converted to a lipid peroxy radical {LO0-} by a"peroxidat€on" reaction with 02. Note the shift of a double bond to form a conjugated diene. Lipid peroxy radicals are highly unstable species which will abstract a hydrogen from a nearby lipid species (LH) in a"propagation" reaction to form a lipid radical ¢.-j plus a lipid hydroperoxide {LOC}ii}. Alternatively the unstable lipid peroxy radical will rearrange to form unstable endoperoxides that break to form alkoxy radicals (LO•), and will eventually °decompose" to shortened oxidized phospholipids, small reactive aldehydes (eg, malondialdehyde) , and reactive alkenals (eg, 4-hydroxynonenal). Also Fe can stimulate the decomposition of lipid hydroperoxides. DETOXIFICATION REACTIONS RADICAL _$CAVENGING GLUTATHIONE PERO%IDASE ALDEHYDE DEHYDROGENASE GLUTATaIONE TRANSFERASE Vitamin E can terminate lipid peroxidation reactions by donating a hydrogen atom {H•} to a lipid radical in a "scavenging reaction. After the enzyme phospholipase cleaves phospholipid hydroperoxides to fatty acid hydroperoxides, this reactive species can be detoxified by the enzyme gfutathlone peroxidase to a fatty acid. alcohol. Several aldehyde dehydrogenase isozymes readily detoxify malondialdehyde and other short chain reactive aldehydes to more stable acids. Glutathione transferase can detoxify 4-hydroxynonenal and other hydroxyalkenals by a GSFH conjugation reaction. 0 i
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Moslen - II Page 33 i SUMMARY 1) Protection against the injurious actions of free radicals is provided by a complimentary complex of * scavenging agents * detoxification enzymes * regenerating processes * export/excretion systems that act within * organelles * cells ~ * extracellular fluids * whole organism. 2) Injury can occur when components of these systems are * overwhelmed by large numbers of free radicals * inadequate due to dietary or other deficiencies * not present or not functional in genetic diseases. 00 w i `~-J CO 0 0 C, ~
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Moslen- Page 20 Do Tissues Vary in Vulnerability to an Antioxidant Deficient Diet? Effects of a Vitamin E Deficient Diet on Tissue Antioxidants and Peroxidation Products at 8 Weeks Tissue a Tocopherol Glutathione 14IDA Lipid Hydroperoxide % Control Diet Brain 50.0 ** 110 107 100 Heart 3.5** 101 113 145* Lung 2.8** 93 79 310** Liver 1.0** 92 116* 80 Kidney 4.6** 113 117* 145** Values are percentages of values in control rats fed a vitamin E sufficient diet *p < 0.05 versus control diet **p < 0.01 versus control diet Tokumaru et aI Free Radical Res 26:169-174, 1997 Note effects vary by tissue, by antioxidant, and by peroxidation product. Brain, which showed a modest depletion of a tocopherol, was spared from a rise in lipid hydroperoxides. In contrast lung, which showed a severe depletion of a-tocopherol, exhibited a three fold rise in lipid hydroperoxides. 0
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Moslen - Nd '3age 28 Antioxidants and Heart Disease - 2 jJ111 Cambridge filter-aglass fiberftlterthat retains 99% of particles larger than 0.1 µm CI' - GAS-PHASE C NO NO R• ROO• CIGARETtE SMOKE , , Z, ROONO, ROONO, \ I Cigarette b Pryor and Stone T AR e -. O~ semiquinone radical on filter Annals NY Acad Sci 686:12-28 1993 , 1 Aqueous cig tar solutions (ACT) 02iM20211-10• A cartoon outlining the separation of cigarette smoke into (a) gas-phase and (b) particulate matter (or tar) by the use of a Cambridge filter. (c) Gas-phase smoke contains both carbon-centered and oxygen-centered radicals that are produced from NO/NOZ reactions with reactive compounds in smoke (such as isoprene). The R' and ROO' radicals can be observed by ESR spin trapping. Tar contains a semiquinone (e), the ESR signal of which can be observed on the filter. This semiquinone can be extracted into aqueous cigarette tar solutions (ACT), as shown in (g), and these solutions yield superoxide and hydrogen peroxide. In the presence of iron, these solutions produce the hydroxyl radical, which can be spin trapped (h). Smoking Status an d F2 - Isoprostanes Levels m 0 ~ 2 000 0 m U w -° 1 00 • nonsmokers O smokers 0 e 1 200 0 ~ CL 0 00 F ~ m c g00~ X'V ~ • 0 ° ° Q 0 I • ss v` o 400 ~ . 0) oo m r • rA lL 0 < w ~ 0 150 300 450 600 CC) 0 c ~ 0 ~ Plasma F2 - Isoprostanes (pmolniter) N Eng J Med 332: 1198-1203, 1995 '
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MosHxas 0 H Page 18 Distribution of Protection Systems Within Cefls ? Vitamin E and GSH R-Carotene Giutathione Transferase Glutathione Peroxidase Aldehyde Dehydrogenase fNDOPLASMIC RETiCULU Catalase Vitamin E and GSM CujZn SOD Glutathlone Transterase Glutathione Peroxidase Metaliothionein PEROXISOMES LYSOSOMES CYTOPLASM -W ~ MiTOCNONDRION LIPID BtLAY£R OF ALL CELLULAR MEMBRANES Vitamin E 6-Carotene mins C and E and GSt! Cu/Zn SOD Glutathione Transferass Gtutathione Peroxidase Aldehyde Dehydrogenase Ferrttin MetaEtothionein Vitamin E and GSH Mn SOD Glutathione Transferase. Giutathfone Peroxldasa Aidehyde Dehydrogenase Distribution of the antioxidants, detoxification enzymes, and transition metal binding proteins that comprise the intracellular protection system within cellular membranes and organelles. Note that the nucleus, with its vital genetic material, is protected by lipid and water soluble antioxidants, by SOD, by glutathione transferase and peroxidase and by :~ metal binding protein. The mitochondrion, where •t}Z is regularly produced by electron transport, is protected by multiple antioxidants, enzymes which detoxify •Oz , H2a2, and products of lipid peroxidation. (Modified from Mosien, 1992).
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Ar~ 2GsH-%,,... Caistase H20 Grutathiane Gfutalikxe pemidase reductase v GSSG...W H20
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Mosten - It Page 22 Why Do Nitroxides Have Therapeutic Potential? • A) Family of stable free radicals compounds. R = H, TEMPO R = OH, TEMPOL R = CO, 4-oxo-TEMPO B) Mimic activity of superoxide dismutase. 1) + 'OZ• + 2H+ -30 + -O + H20Z + pZ C. TEMPO Reported to Protect Isolated Perfused Hearts Against Detrimental Effects of ]schemia (5 min) Followed by Reperfusion ~ ventricular Cibrillation ~ ventricular tachycardia p normal sinus rhythm 0 other rhythm 3001 600 500 v y 300 s c~ QJ L ~ ,~ 200 ~ N 0 : Control Ethanol 19M0 _ _ _ Treatment ~ 1004 0 Gelvan et al, ~ 991 '' ~ CPJ Controd Co . t........ i .......................... TEAM+p 10 20 Reperfusion time (min) 30
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Moslen - tg Page 26 Why Does Fe Influence ROS Formation? Could Antioxidants Help? 0 Fe2+ /Cu' + Fe3+ /Cuz+ e e e 02 H+' 02 H+'- H202 H+ 'OH superoxide hyidrogen hydroxyl radical peroxide radical X LOOH + Fe2+ /Cu1+-~10 Fe3+ /Cu2+ + OH + LO• LOOH + Fe3+ C 2+On l+ 2+ + / u Fe /Cu + H + LOO• Is Plasm wi a Antioxid th Hemoc ants in Adults hromatosis Controls N=15 Hemochromatosis N=15 p ~ w Vitamin E 7.24 5.91 0.001 w OD Vitamin A 2.46 1.78 0.001 C) C) Witamin C 89.1 51.3 0.013 tr ~ TBARS 0.46 0.59 0.045 units are gmol/l Young et aY ~ Free Rad. Biol. Med. 16: 393, 1994
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Moslen Page 12 t.IPID PEROXiQATiQN REACTF8N5 NVYY~~ Polyunsaturated fatty acid of a membrane phospholipid is converted to lipid radical by an "initiation" reaction with •OH. The lipid radical is converted to a iipid peroxy radical (LOO •) by a"peroxidation" reaction with O.. Note the shift of a double bond to form a conjugated diene. i_ipid peroxy radicals are highly unstable species will abstract a hydrogen from a nearby lipid species in a"propagation" reaction to form a iipid radical (L• ) plus a iipid hydroperoxide (LOOH). Alternatively the unstable iipid peroxy radical will o~ rearrange to form unstable endoperoxides that break to form alkoxy radicals (LO •), and will eventually a= "decompose" to shortened oxidized phospholipids, small reactive aldehydes (eg, maiondiaidehyde) , and reactive alkenals (eg, 4-hydroxynonenal). Also Fe can stimulate : 0othe decomposition of iipid hydroperoxides. 7 cc w 0 ~ L~t
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Moslen Page 9 ! i OR Macromolecular Targets of ROS DNA CH3 HO• + HO. niy T ~}-OH \ N H2N ~ HO• R'O OR CHpOH Strand Scission w w ~ Co 0 C:) ~
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Moslen - lI Antioxidants and Heart Disease - 5 Page 31 • Effects of an 84 hr Cessation of Smoking on Antioxidant Status in Plasma and LDL Antioxidant Plasma LDL % Change a Tocopherol y Tocopherol (3 Carotene Lycopene Total Vitamin C i J. Nutri. Biochem 7:29,1996 00 W ~ 00 O 0 Cri t" Values are means tSE for 6 male smokers * p < 0.05
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Antiaxidants and Heart Disease - 4 ~ Ib4oslen - Page 30 Effects of Antioxidant Vitamins on Death of Iowa Women from Coronary Heart Disease i.25 1.25 Vit E suppteaaaea8.s ~ p= 0.39 0.95 s 1.09 s 0.76 - Vit E diet only p = 0.004 t i I ~ i I 1 2 3 4 5 Lowest Quintile of Antioxidant Int'a.ke 1.00 Highest Effects of Antioxidant Vitamin E-Rich Foods on Death of Iowa Women from Coronary Heart Disease .---~,~ . Margarine . p=tl.{I43 s i Mayonnaise g = 0.469 00 CNI 01-1 M O Cn t1'~ I I I -A ~ 1 2 3 4 ~ Lowest Extent of Intake Highest Kushi et aI, N.Engl J Med 334: 156, 1996
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Moslen Page 17 Synthesis of NO- ! Arginine L-Synthetase enhanced by arginine inhibited by L-NAME or L-NMA Effects on Ischemia-Reperfusion Injury in Rats treated with modulators of Arginine L-Synthetase L-NMA LUNG L•NAME a L•Sr0lnln. POSIRw Control 0.0 0.5 1.0 VASCULAR PERMEABILITY INDEX Figure from Seekamp et ai, 1993
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Moslen :1 Page:14 READING LIST Bacon BR, Britton RS. The pathology of hepatic iron overload: A free radical-mediated process? Hepatology 11:127-137, 1989. Benidick A, Machline LJ, Scandurra O. The antioxidant role of Vitamin C. Free Radicals Biol Med 2:419, 1986. Beutler E. Glucose-6-phosphate dehydrogenase deficiency. NEngJMed 324:169-174, 1991. Boesch JS, Lee C, Lindal RG. Constitutive expression of class 3 aldehyde dehydrogenase in cultured rat corneal epithelium. The JBiological Chem 271:5150-5157, 1982. Brown KM, Morrice PC and Duthie GG. Erythrocyte vitamin E and plasma ascorbate concentrations in relation to erythrocyte peroxidation in smokers and nonsmokers: dose response to vitamin E supplementation'•2. Am J Clin Nutr 65:496-502, 1997. Buettner GR The pecking order of free radicals and antioxidants: Lipid peroxidation, a-tocopherol, and ascorbate. Arch Biochem Biophys 300:535-543, 1993. Chang LY, Slot JW, Geuze HI, Crapo JD. Molecular immunocytochemistry of the CuZn superoxide dismutase in rat hepatocytes. J Cell Bio1107:2169-2179, 1988. Christen S, Woodall AA, Shigenaga MK, Southwell-Keely, PT, Duncan MW and Ames BN. y- Tocopherol traps mutagenic electrophiles such as Nox and complements a-tocopherol: Physiological implications. Proc Natl Acad Sci 94:3217-3222, 1997. Diaz MN, Frei B, Vita JA, Keaney JF. Antioxidants and Atherosclerotic heart Disease. NEngJMed 337:408-416, 1997. Esterbauer H, Schaur RJ, Zollner H. Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related alehydes. Free Radicals in Biol Med 11:81-128, 1991. Frank L, Iqbal J, Hass M, Massaro D. New "rest period" protocol for inducing tolerance to high OZ exposure in adult rats. Am JPhysio1257:L226-L231, 1989. - Gelvan D, Saltman P, Powell SR Cardiac reperfixsion damage prevented by a nitroxide free radical. Proc Natl Acad Scf 88:4680-4684, 1991. Gollan JL. Copper Metabolism, Wilson's disease, and hepatic copper toxicosis. IN: Hepatology A textbook of Liver Disease. Zakin D, Boyer TD, eds. 2nd ed W.B. Saunders, Philadelphia, 1990; pp _ 1249-1272. . 0 Griffith OW, Meister A. Glutathione: Jnterorgan translocation, turnover, and metabolism. Proc nw" •
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Moslen Page 7 0 Production of ROS by Ischemia-Reperfusion . Q ~ ~ = ATP mitochondrial .H injury I ~ . "leaky" electron transport 1 REPERFUSION 02 AMP Xanthine ~ Dehydrogenase Adenosine 1 Inosine ~ Hypoxanthine REPERFUSION Urate + 02 w w w CO i ° t:D CJ. OD Xanthine Oxidase
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~ Wrecking Crew of Reactive Oxygen Species (ROS) Moslen Page 10
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Moslen Page 1 Free Radicals Mary Treinen Moslen, Ph.D. William C. Levin Professor of Environmental Toxicology Director, Toxicology Training Program ~ University of Texas Medical Branch Galveston, Texas Maty'Freinen Moslen was born in Iowa, received a BA in chemistry from Vassar Cottege, a MS in scientific communication from Boston Universrty, and a PhD from LTTIviH. Her research interests are cell injury and toxicant effects on bile formation. She is a member of the Society of Toxicology and the Socsety of Investigative Pathotogy. Her service on national committees included membership on the NIH Toxicology Study Section. She has served on the ediiorial6oards of several journals and as an associate editor of Toxicology and Applied Pharmacology. tr3 rr~ c~ 0 C) CN r.5
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masten Page 8 Macromolecular Targets of ROS LIPIDS 02 H H s0-O Hd-{} Macromolecular Targets of ROS PRt}TEINS 9 Vulnerable sites include cysteine and histidine Examples Ca-ATPase Metal Binding Proteins Superoxide Dismutase Catalase Glucose-6-Phosphatase Many Other Enzymes co w
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Moslen - II Page 37 van den Berg JJM, Op den Kamp JAF, Lubin BH, Kuypers FA. Conformational changes in oxidized ~ phospholipids and their preferential hydrolysis by phospholipase AZ: a monolayer study. Biochemistry 32:4962-4967, 1993. Yoshikawa T, Furukawa Y, Murakami M, Takemura M, Kondo M. Effects of Vitamin E on D- galactosamine-induced or carbon tetrachioride-induced hepatotoxicity. Digestion 25: 222, 1982. ! 00 w w ! CO a CD ON I
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Moslen - II Page 35 ! Acad Sci 76:5606, 1979. Hafez M, Amar ES, Zedan M, Hammad H, Sorour AH, El-Desouky ESA, Gamil N. Improved erythrocyte survival with combined Vitamin E and selenium therapy in children with glucose-6- phosphate dehydrogenase deficiency and mild chronic hemolysis. JPediatr 108:558-561, 1986. Halliwell B. Commentary: Antioxidant characterization. Pergamon 49:1341-1348, 1995. Halliwell B, Gutteridge JMC, Cross CE. Free radicals, antioxidants, and human disease: Where are we now? JLab ClinMed 119:598-620, 1992. Harris ED. Regulation of antioxidant enzymes. FASEB 6:2675-2683, 1992. Holley AE, Chesseman KH. Measuring free radical reactions in vivo. British Medical Bulletin 49:494-505-, 1993. Hooper C. Free radicals: Research on biochemical bad boys comes of age. JNIHRes 1:102-106, 1989. Ishikawa T, Esterbauer H, Sies H. Role of cardiac gllutathione transferase and of the glutathione S- conjugate export system in biotransformation of 4-hydoxynonenal in the heart. J Biol Chem . 261:1576, 1986. Keshan Disease Research Group of the Chinese Academy of Medical Sciences, Beijing. Epidemiological studies on the etiologic relationship of selenium and Keshan disease. Chinese Med J 92:471-482, 1979. Kukovetz EM, Bratschitsch G, Hofer HP, Egger G, and Schaur RJ. Influence of age on the release of reactive oxygen species by phagocytes as measured by a whole blood chemiluminescence assay. Free Rad Biol Med 22:433-438, 1997. Kushi LH, Rolsom AR, Prineas RJ, Mink PJ, Wu Y, Bostick RM. Bietary antioxidant vitamins and death from coronary heart disease in postmenopausal women. NEngJMed 334:1156-1162, 1996. Laenstein R, Epp 0, Huber R, Wendel A The structure of glutathione peroxidase from bovine red blood cells. IN: Selenium in biology and Medicine. AVA Publishing Co 1981; Westport, P33. Liebler DC, Kaysen KL, Kennedy TA. Redox cycle of Vitamin E. Hydrolysis and ascorbic acid dependent reduction of 8a-(alkydioxy) tocopherones. Biochemistry 28:9772, 1989. W Lombard M, Bomford AB, Polson RJ, Bellingham AJ, Williams R. Differential expression of ~ transferring receptor in duodenal mucosa in iron overload: Evidence for a site-specific defect in 05 ~ genetic hemochromatosis. Gastroenterology 98:976-984, 1990. f~ tJ't 1t.9
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.rausien Page 14 Consequences of ROS Membrane Dysfunction Cytotoxicity •0= , •OH, R• ~---r~ Li id ~ ~ p PeroAdation --~ ~ AldehYdes ~ ~ Alkenals } Oaddized ins Prote Olddized DNA Protein Dysfunction ~ Atherosclerosis J Cataracts ~ Other Health Impaired Problems DNA Repair f Teratology Mutation -~~ { Neoplasia . *I i Oaddized
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Moslen Page 5 0 0 CELL INJURY Lipid peroxidation Protein damage DNA damage 00 w 04 co cn) CN ch How Are Reactive Oxygen Species Formed ? - - - • ~ - - ~ -, ~ - ~ , - . - - - - ~ PMNs, macrophages f -~- Inflamm88on. - PMNs, xanthine ox,dase;-P gfterischemia ' _,'Reperf.:sion injury : cyclic redox reactions Chemical toxicity ~ Mixed function oxidation ` lonizing ~ radiation •O2 HZOZ •OH REACTIVE OXYGEN SPECIES
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Moslen Page 15 Oxidized Metabolites of Nitrogen: Nomenclature Symbol Name Comments NO' nitric oxide free radical NOZ• nitrogen dioxide free radical, nitrosating agent N20 nitrous oxide anesthetic (laughing gas) Nz04 dinitrogen tetroxide nitrosating agent (dimeric NOZ' ) NOZ - : NO3 - nitrite produces NO' at acidic pH nitrate stable ion ~ cl~
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Mosien - Iy Page 3 6 Hughes H, Mathews B, Lenz ML, Guyton JR. Cytotoxicity of oxidized LDL to porcine aortic smooth muscle cells is associated with the oxysterols 7-Ketocholesterol and 7-Hydroxycholesterol. Arterioscler Thromb 14:1177-1185, 1994. Mitchell DY, Petersen DR The oxidation of a-[3 unsaturate aldehydic products of lipid peroxidation by rat liver aldehyde dehydrogenases. ToxAppd Pharmacot 87:4Q3-414, 1987, Morrow 7D, Frei B, Longmire AW, Gaziano M, Lynch SM, Shyr Y, Strauss WE, Qates IA, Roberts LI. Increase in circulating products of lipid peroxidation (F2-Isoprostanes) in smokers. NEngJMed 332:1198-1203, 1995. Moslen MT, Smith CV editors, Free Radical Mechanisms of Tissue Injury. Baton Rouge: CRC Press, Protection against free radical-mediated tissue injury. P 203-215, 1992. Moslen MT, Armstrong D, editors. Free Radicals in Diagnostic Medicine: A Systems Approach to Laboratory Technologies, Clinical Correlations, and Antioxidant Therapy. New York: Plenum Press, Reactive oxygen species in normal physiology, cell injury and phagocytosis. P 17-27, 1994. Oberley TD, Oberley LW, Slattery AF, Laurichner L7, E1wel17II. Immunohistochemical localization ofantioxidant enzymes in adult Syrian hamster tissues and during kidney development. Am JI'athoi 137:199-214, 1990. Ogihara T, Miyake M, Kawamura N, Tamai Ii, Kitagawa, Mino M. Tocopherol concentrations of leukocytes in neonates. Ann NYAcad Sci 57t):487, 1989. Pietrangelo A, Rocchi E, Casalgrandi G, Rigo G, Ferrari A, Perini M, Ventura E, Cairo G. Regulation of transferrin, transferrin receptor and ferritin genes in human duodenum. Gastroenterology 102:8t12-809, 1994. Pryor WA and Stone K. Oxidants in cigatette smoke: radicals, hydrogen peroxide, peroxynitrate, and perorcyrritrite. In: Tobacco Smoking and ATutrition: Influence ofl+Iutrition on eniy of Sciences, edited by 7. Diana and W.A. Pryor. New York: New York Academy of Sciences, f,85:12-28, 1993. Robbins SL, Cotran RS, Kumar V. Robbins Pathologic Basis of Disease. 4th ed, Philadelphia: W B Saunders Co; 1989.Stemmel W, Reidel I3D, Niederau C, Strohmeyer. Pathogenesis of genetic haemochromatosis. Europ J Clim Imwst 23:321-329, 1993. Roberts L7, Moore KP, Zackert WE, Qates IA, Morrow ID. Identification of the major urinary metabolite of the FZ-isoprostane 8-Iso-prostaglandin E in humans. J of Biological Chem 271:20617-20620, 1996. Stemmel W, Reidel HID, Niederau C, Strohmeyer G. Pathogenesis of genetic haemochromatosis. Europ,I Ctin Invest 23:321-329, 1993. 00 !
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GRIPPO A • Receptors in Toxicological Mechanisms r Joseph F. Grippo Hoffmann-La Roche Inc. Department of Toxicology and Pathology 00 Nutley, New Jersey w w ,~ a> i r-) CD - CC7 .r~
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Effects of Peroxidation on a Membrane ? 0 Membrane surface proteins ~ Disulfide crosslinking 1 H 0 C~GJJ v SS'10/ln O/~ 0 I IliH^i;i"°ell from oxidized fatty acids R AIosleu Page 13 Transmembrane glycoprotein if Protein strand scission Malondialdehyde released,w~ oxidation _d S CH3 ` crosslinking ~ Fatty acid Orderly arrangement of membrane phospholipids and proteins in a representative cell membrane (top diagram) is disrupted by free radical initiated lipid peroxidation reactions (bottom diagram). Alterations to phospholipids include shortened oxidized fatty acids and the release of ! small reactive aldehydes such as malondialdehyde which can crosslink lipids or proteins. Alterations to proteins include strand scissions and inappropriate disulfide crosslinks. (Diagrams from Robbins et al, 1989)
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6RIPPOII-13 i SATURATION KINETICS P-4 ~ 75~ 0 ~ ~ °' S0 4" ~ b ~ 25 a p ~ 0 4 8 12 Free ligand (nanoMolar) s r 0 0 0 KD= 1 nM ~- -
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GRIPPO 1-9 G-PROTEN LINKED RECEPTORS fNTRACELLULAR .. . }'tiGL ! ttH2 EXTRACELLULAR ! • irrOGC~C~ i
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~s~uNCL Page 22 ' REFERENCES G.R. Buettner. The pecking order of free radicals and antioxidants: lipid peroxidation, a-tocopherol, and ascorbate.' Arch Biochem Biophys 1993;300:535-543. H. Esterbauer, R.J. Schaur, H. Zollner. 'Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related alehydes.' Free Radicals in Biol Med 1991;11:81-128. C. Hooper. Tree radicals: Research on biochemical bad boys comes of age.' J NIHRes 1989;1:102-106. Moslen MT. ; Moslen MT, Smith CV, editors. Free Radical Mechanisms of Tissue Injury. Baton Rouge: CBC Press, 1992; Protection against free radical-mediated tissue injury. p. 203-15. Moslen MT. ; Armstrong D, editors. Free Radicals in Diagnostic Medicine: A Systems Approach to Laboratory Technologies, Clinical Correlations, and Antioxidant Therapy. New York: Plenum Press, 1994; Reactive oxygen species in normal physiology, cell injury and phagocytosis. p. 17-27. V.L. Kinnula, J.D. Crapo, K.O. Raivio. Biology of Disease: Generation and disposal of reactive oxygen metabolites in the lung' LaboratoryIrrvestigation 1995;73(1):3-19. G.R. Buettner. The Pecking order of free radicals and antioxidants: Lipid peroxidation, a-Tocopherol, and ascorbate.' Archtves of Biochemistry and Biophysics 1993;300(2):535-543. B. Halliwell, J.M.C. Gutteridge, C.E. Cross. Tree radicals, antioxidants, and human disease: Where are we now?' J Lab Clin Med 1992;119(6):598-620. A.E. Holley, K.H. Cheeseman. 4vleasuring free radical reactions in vivo.' British Medtcal Bulletin 1993;49(3):494-505. B. Halliwell.'Commentary: Antioxidantsharacterization.'Pergamon 1995;49(10):1341-1348. H. Wiseman, B. Halliwell: 'Review Article: Damage to DNA by reactive oxygen and nitrogen species: role in inflammatory disease and progression to cancer.' Biochem J. 1996; 313: 17-29. J.D. Marrow, K.E. Hill, R. F. Burk, T.M. Nammour, K.F. Badr, L.J. Roberts II: 'A series of prostaglandin F2-like compounds are produced in vivo in humans by a non-cyclooxygenase, free radical-catalyzed mechanism' Proc. Natl. Acad Sci. 1990; 87: 9383-9387. A. Seekamp, M.S. Mulligan, G.O.Till, P.A. Ward: 'Requirements for Neutrophil Products and L- Arginine in ischemia-reperfusion injury' American Journal of Pathology 1993; 142 (4): 1217- 1226. M.B.Grisham. 'Reactive metabolites of oxygen and nitrogen in biology and medicine' R.G. Landes Company 1992.
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GRIPPO 1-4 0 LIGAND-RECEPTOR THREE DfMENSIONAL INTERACTION 0
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GRIPPO C 9 RECEPTORS IN TOXICOLOGICAL MECHAIVISMS I. INTRODUCTION A. Lock and key B. Three-dimensional representation of receptor binding sites C. Classes of Receptors 1) Agonist-gated channels 2) Agonist-regulated enzymes 3) G-protein-coupled receptors 4) Nuclear receptors II. ANALYSIS OF LIGAND-RECEPTOR INTERACTIONS A. Competitive binding of Ligands B. Saturation Kinetics C. Scatchard analysis D. Agonists and antagonists i III. THE NUCLEAR RECEPTORS A. General structure B. Mechanism of action C. Transient transactivation assay for nuclear receptor function IV. ACTION OF NUCLEAR RETINOIC ACID RECEPTORS A. Retinoid-induced teratogenesis B. Nuclear retinoic acid receptors C. Model for retinoid action D. Receptor-selective retinoids E. Receptor-selective physiology rxs 04 tI.f ~ C:) tcs bN
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GRIPPO B ~ Dr. Joseph F. Grippo, Department of Toxicology and Pathology, Hoffmann-La Roche Inc., Nutley, New Jersey, 07110 Dr. Grippo is a Research Investigator in the investigative Toxicology group within the Department of Toxicology and Pathology at Hoffmann-La Roche. His major research interest involves determining specific pathways of gene expression that are mediated by interaction of retinoids with their nuclear receptors. His laboratory focuses on examining retinoid receptor-mediated alterations in the expression of genes that are important for early mouse development. Dr. Grippo received a B.A. in Biology at )ohns Hopkins University and a Ph. D. in Pharmacology at the University of Michigan in Ann Arbor, Michigan. In Ann Arbor, Dr. Grippo worked with Dr. Pratt to define endogenous factors that influence the ability of glucocorticoids to bind to their receptors. Afterwards, Dr. Grippo did postdoctoral work with Dr. Gudas at the Dana Farber Cancer Institute in Boston, Massachusetts where he began to work on the molecular mechanisms of retinoid action in F9 teratocarcinoma cells. ~ In 1989, he joined Huffmann-La Roche and has developed a program to understand molecular mechanisms of retinoid receptor biology. Dr. Grippo received the Roche Research and Development Award in 1991 for his work in determining that 9-cis retinoic acid is the proximate ligand for the retinoid receptor subfamily, RXRs. c.t0 w c~t ~ 05 C`J G10 ~
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GRIPPO IV-26 0 00 C14 . C~j OD 0 /r- mI ^ ~ W
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GRIPPO I-10 ADRENERGIC RECEPTOR ACTIVITIES Q a_AR ~- ~/ ~ GTP GDP _ _ S AR TT~ GDP GTP PROTEIN KINASE A PIP, a i ADENYLYL CYCLASE cAMP ATP CELLULAR RESPONSES PROTEIN KINASE C IP GTP GDP GG h ~ jJ = PLC ~ a,AR 1I n I DAG Cb f_~d W ~ On d C.7 \0 CYt
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GRIPPO 11-12 Competitive Inhibition of Radioligand Binding 0 100 m is E 50 ---- ~ i ~ IC50 i ~ L I ~ C. I 0 1 10 100 1000 Concentration (Log Scale) ~ ~ C-j ~ ~ ~ ~
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General Structure of Nuclear Receptors A B C D IfEl F DNA Binding Ligand Binding aQ Go RM
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! ! ! RXRs INTERACT WITH OTHER NUCLEAR RECEPTORS TO FORM FUNCTIONAL HETERODIMERS RXR Thyroid Hormone Receptors TRE RXR Vitamin D Receptor CV~~ RXR RARs 17 tLGG9229
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00~08Zi8 a A 0 -4 m z 3 0 l3 4A BOUND ff moles/ mp protein x 10-Z1 t - N W A (]I pt rn 1 m1 •1 D mF i .~ t3H] RA BOUND (t moles x 10-Z) -O - N _ W A__N al 0 N 0 A 0 OI 0 m O+ \Q \ (9 \ ^1 a E 3H] RA BOUND (f moles/mq protein x 10-z1 •
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GRIPPO 111-24 Transactivation: Ligand-dependent expression of reporter gene. i Response Element Promoter Reporter Gene nding DNA Binding Ligand Bi Domain Domain Reporter cDNA Receptor cDNA / Receptor cDNA Transcription/ Reporter cDNA Translation Ligand+ /Nucleus / TrcnSCricticll.'TranSja'icn I ~ 1 Measurable Levels of Reporter Protein O I 0 w
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GRIPPO 111-23 NUCLEAR RECEPTORS BIND TO DNA AS DIMERS GA N GR N iAGAACAI- 3 -;TGTTCTi 5' • RXR 3' RAR AGGTCA I- 5 -;AGGTCA I 5' 3' I/V ~ w ~ co CD . 1 Y~ C) Wy
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GRIPPO IV-25 0 Phenotype of Retinoic Acid Induced Teratogenesis Treated Control OOH i Phenotype of retinoid-induced teratogenesis. C57BU6 dams received either 60 mg/kg of retinoic acid or 5 ml/kg of Tween 80 vehicle on day 8.5 pc. Embryos were recovered on day 17.5 pc. The control embryos is on the right 3 and the retinoic acid treated embryo is on the left. The retinoic acid treated ~ embryo exhibits terata associated with high doses of retinoic acid such as C'4 retarded growth, open eyes, spina bifida aperta, and the absence of ears, CYJ lower jaw and tail. ~ _~
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Two Families of Retinoid Receptors and Receptor Subtypes alpha B D beta 97% E F 90% gamma 141 '0 D s40i 97% 462 448 458 ~ vS ` k~,~ C~/,q C ~~ z, ~~- RARs 95% RXRs D 86% 467 410 463 ~
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Levels of Complexity Isomer Selectivity sctas22s i all trans-Retinoic acid 9-cis Retinoic acid 7>M 'd- ~ Response Elemen Selectivity 0 0
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RAR D Two Families of Retinoid Receptors RXR 462 o \\\\©\\\\%o~ 61% 27% 467
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GRIPPO 111-22 • SPACING DETERMINES RECEPTOR SELECTIVITY AGGTCA AGGTCA co w w CO 0 ~ 0 ~
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. S Patterns of Binding of Retinoids to RARs 0 0 8 7 0 OIL 109228 - Log Concentration (M) 5 0
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HOW RETINOIDS INDUCE A CASCADE OF GENE EFFECTS 9LGQ9228
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GRIPPO B ~ RETINOIDS AND TERATOGENESIS: MOLECULAR APPROACHES AND TECHN/QUES (Lecture Outlinel 1. INTRODUCTION A. B. Molecular and cellular approaches to understanding developmental toxicity: Retinoid-induced teratogenesis as a model system Mechanism of retinoid action 1. Nuclear retinoic acid receptors 2. Retinoid action at the gene level C. 1. Retinoid-responsive target genes Homeobox genes II. ISOLATION AND CHARACTERIZATION OF RETINOID-RESPONSIVE DEVELOPMENTALLY-IMPORTANT GENES IN EMBRYOS 0 A. Identify sensitive window of retinoid-induced teratogenesis B. C. D. Isolate and characterize retinoid-responsive genes in embryos Characterize retinoid-responsive genes in embryos by in situ hybridization Characterize retinoid response elements using lac Z fusion genes in transgenic animals III. ALTERATION OF THE EXPRESSION OF RETINOID-RESPONSIVE DEVELOPMENTALLY-IMPORTANT GENES IN TRANSGENIC ANIMALS A. Loss of function 1. Homologous recombination B. Gain of function 2. Ectopic expression C. Analysis of associated phenotypes co w t~.I ~ 10 CD hJ CrJ
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Retinoid Receptors Mediate Both Therapeutic and Toxic Actions RETINOIDS Receptor Subtypes Therapeutic Action Toxic Action Goal: Identify receptor-specific physiologies and design receptor-specific ligands. LIG08228
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GRIPPO IV-34 • RETINOID TOXICITY IN MICE LACKING RAR GAMMA • RARt.-1- N 'L RARI•+1- I" RARf+/+ + +RA +RA ff +RA ~ •
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GRIPPO A ~ Dr. Joseph F. Grippo. Department of Toxicology and Pathology, Hoffmann-La Roche Inc., Nutley, New Jersey, 07110 Dr. Grippo is a Research Investigator in the Investigative Toxicology group within the Department ofToxicologyand Pathologyat Hoffmann-La Roche. His major research interest involves determining specific pathways of gene expression that are mediated by interaction of retinoids with their nuclear receptors. His laboratory focuses on examining retinoid receptor-mediated alterations in the expression of genes that are important for early mouse development. Dr. Grippo received a B.A. in Biology at Johns Hopkins University and a Ph. D. in Pharmacology at the University of Michigan in Ann Arbor, Michigan. In Ann Arbor, Dr. Grippo worked with Dr. Pratt to define endogenous factors that influence the ability of glucocorticoids to bind to their receptors. Afterwards, Dr. Grippo did postdoctoral work with Dr. Gudas at the Dana Farber Cancer Institute in Boston, Massachusetts where he began to work on the molecular mechanisms of retinoid action in F9 teratocarcinoma cells. i In 1989, he joined Hoffmann-La Roche and has developed a program to understand _ molecular mechanisms of retinoid receptor biology. Dr. Grippo received the Roche Research and Development Award in 1991 for his work in determining that 9-cis retinoic acid is the proximate ligand for the retinoid receptor subfamily, RXRs. tXO cr+ t~t ~ ~ ~
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I - 1 ~ Phenotype of Retinoic Acid Induced Teratogenesis Treated Control OOH • Phenotype of retinoid-induced teratogenesis. C57BV6 dams received either 60 mg/kg of retinoic acid or 5 ml/kg of Tween 80 vehicle on day 8.5 pc. Embryos were recovered on day 17.5 pc. The control embryos is on the rightw and the retinoic acid treated embryo is on the left. The retinoic acid treated C,~',, embryo exhibits terata associated with high doses of retinoic acid such as ~ retarded growth, open eyes, spina bifida aperta, and the absence of ears, ~ lower jaw and tail. Na .fz~
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GRIPPO 35 • REPERENCES 1. The Pharmacological Basis of The.apeutics (1990) edited by Alfred Goodman 2. Gilman, Theodore W. Rall, Alan S. Niles 2-nd Palmer Taylor, Pergamon Press, New York. Sargent, P. The diversity of Neuronal Nicotinic Acetylcholine Receptors. (1993) 3. Annual Review of Neuroscience, 16, pp. 403-443. Kobilka, B. Adrenergic Receptors as Models for G Protein-Coupled Receptors. 4. (1992) Annual Review of Neuroscience, 15, pp. 87-114. Bertolino, M. The Central Role of Voltage-Activated and Receptor-Operated 5. Calcium Channels in Neuronal Cells. (1992) Annual Review of Pharmacology and Toxicology, 32, pp. 399-421. Yuen, P. S. T. and Garbers, D. L. Guanylyl Cyclase-Linked Receptors. (1992) 6. Annual Review of Neuroscience, 15, pp. 193-225. Beato, M. Gene Regulation by Steroid Hormones. (1989) Cell, 56, pp. 335-344. 0 7. Lohnes, D., Kastner, P., Dierich, A., Mark, M., Le Meur, M. and Chambon, P., Function of Retinoic Acid Receptor y in the Mouse. (1993) Cell, 73, pp. 643- 658. rn tA .~ IJ t~
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I - 8 ! Temporal and Spatial Expression of Genes in Development db Fully Mature Fetus co trw ~ ~ ~ ~ t~
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RETINOIDS AND TERATOGENESIS: MOLECULAR APPROACHES AND TECHNIQ UES Joseph F. Grippo, Ph.D. Hoffmann-La Roche Inc. Department of Toxicology and Pathology Nutley, New Jersey
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RETINOID BINDING PROTEINS ~ ~
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I - 3 Family of Retinoic Acid Receptors RAR alpha 'Q'10 RAR beta IE © D 462 0 0 ~\\\\~©~\\\\~i~i RAR gamma 0 0 RXR alpha 0 © RXR beta 448 458 Number of Isoforms 7 3 7 230 D 467 1 171 [E B7 C 0 E F 410 1 95% 92% RXR gamma 229 B 0 ~ : a ~\\\N©\\\\\\~`i@ i 0 0 \\\\\\©IR 95% 86% 463 1
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General Structure of Nuclear Retinoid Receptors A 0 DNA Binding D Ligand Binding F a
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HOW RETINOIDS INDUCE A CASCADE OF GENE EFFECTS 6Z108228 0 9
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'
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New Levels o?Complexity all trans-Retinoic acid 9-cis Retinoic acid COOH Ligand -,MM 4~ W Binding 11111 "Ligand Domain I Binding DNA Binding Domain Domain ! 8ZL08228
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ISOLATE AND CHARACTERIZE RETINOID-RESPONSIVE GENES DURING THE SENSITIVE PERIOD • 0 ~
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Homeobox Genes Cluster on Four Mouse Chromosomes M Posterior Late , Low RA response a Anterior 00~ Early High RA response L2L08228.
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II - 8 CHARACTERIZE RETINOID-RESPONSIVE GENES IN EMBRYOS BY IN SITU HYBRIDIZATION
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II - 4 0 Overexpression of the Antennapedia Homeobox Gene
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CHARACTERIZE THE RETINOID-SENSITIVE TERATOGENIC WINDOW IN NIICE ! w ~ ~ ca~ ~s
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9 CHARACTERIZE THE TERATOGENIC POTENTIAL OF RETINOID-RESPONSIVE GENE PRODUCTS USING TRANSGENIC ANIMAL MODELS. TRANSGENIC ANIMAL MODELS 1) Loss-of-Function Mutation: with Hox 1.6. Homologous Recombination 2) Gain-of-Function Mutation: Overexpression of Hox 1.6. 0 (20 ~ w CD V% \y
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General Structure of Homeobox Proteins COOH Variable Region Conserved Homeodomain co w. W CO ~
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Rhombomere Transformation in Mouse Hindbrain RAJHoxa-1 kscOR22a
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9 Ectopic Expression of Hox 1.6 Human p-actin promotor & intron Hox 1.6 SV40 coding sequence pA 1 kb Number o embryos transfere f Age when examined d Number of mice born or embryos obtained Number of transgenic Number of malformed 136 after birth 14 0 0 670 9.5 p.c. 156 17 15 0 €7 s 10 32.2 a
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II - 14 B-GALACTOSIDASE REPORTER SYSTEM IN TRANSGENIC MICE 9 5' REGULATORY ELEMEN78 B-GALACTOSIQ4SE CODUVG REGION POLY A B-GALACTOSIDASE + X GAL = BWE CHROMOPHORE 3' cm
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CHARACTERIZE RETINOID RESPONSE ELEMENTS USING LAC Z FUSION GENES IN TRANSGENIC ANIMALS 0
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Expression of P-Galactosidase by Construct A t The developing hindbrain is segmented into rhombomeres (rl-r8) as schematically illustrated. The transgene is prominently expressed in r2 from 8.5-11.5 days p.c.. exhibiting a more anterior boundary of expression than endoaenous Hox 1.6. During this time a lower level of expression is seen in r4 and the somites. 0
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1' A AU
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GENOTOXICOLOGIC MECHANISMS DAVID J. BRUSICK
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. Is B C co w w CO 0 ~ • Expression of P-Galactosidase by Construct D ,At In rnr hybridization of Hv.r 1.6 in a sagittal section from an 8.5 da p.c. embryo shows the normal expression pattern at that stage. ( B.) Construct D directs the transgene expression in a pattern more closely resembling that of endogenous Hox 1.6. suggesting that a major Hos 1.6 re:ulatorv element is located down-stream of the gene (within the 2.6 k 3' traementt. (C) The transsene starts to express in the posterior resio of the emhrvo at 7.5 davs p.c. in mouse embryo, which also agrees with that of endot*enous Hoz 1.6.
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0 4 ;i i I 1 ! 1 1 I I r ) I l~ 14) 0
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Retinoic Acid Hoxa-1 gene Normal and abnormal SSlos228 development
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Effects of RA on Transgene Expression (A) Construct A-generated transgenic mice: Retinoic acid represses the expression of the transgene in ncombomere 2 of the hindbrain of a 8.5 days p.c. embryo (embryo on the left). (B) Construct D-generated trunsgenic mice: Retinoic acid produces an increase in the transgene activity (embryo on the lower left). •' 0
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Embr o Forebrain Hindbrain Neuropore Development Normal y truncation malformation unclosed retardation E91-62A-7 + + E91-62A-8 + + + E91-62B-9 + + E91-6213-11 + E91-62B-15 + E91-73-8 + E91-73-9 + + E91-73-12 + E91-76-2 + E91-76-3 + + E91-76-12 + + E92-1-5 + E92-1-11 + E93-3-2 + E92-3-3 + E92-3-4 + + E92-3-9 + Total (N-17) 7 6 6 4 2 9r~ . L 029 2 29
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Gain-of-Function Mutation of Hox 1.6 A Human P-acnn promotor & imron Expression veavtor Fertilized egg Tranegenio maaa SV~q IM Fig. 6. (A) The ectopic expression vector is made by placing the Hox 1.6 coding sequence under control of a human (i-actin promotor. The DNA is injected into pronuclei of fertilized eggs, which are then transferred into the oviducts of recipient females to develop. (B) The p-actin promotor-driven Hox 1.6 is expressed everywhere in a 9.5 days embryo (left), whereas the endogenous gene is expressed only posteriorly in neural tube and mesoderm (right). (C) The transgenic mouse embryo shows malformation in the developing midbrain (left), as compared with a normal embryo (right) at the same stage. !
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833801 43 0 1*
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• MECHANISMS OF MUTATION David Brusick Hazleton Washington Abstract Data from animals and humans suggests that genetic damage plays an important role in the initiation of toxicity. The current array of tests used in genetic toxicology may not reliably detect all of the genotoxic events which occur in humans, and therefore, may not provide a reliable assessment of the potential hazard from chemical exposures. New types of tests, which are able to measure mutations as well as ~ define the mechanisms of formation at the molecular level, are available and their use can improve the understanding of mutagenic mechanisms and the relationship between genetic damage and toxicity in humans. A review of genetic damage and its role in human toxicity will be used to demonstrate the relevance of some of these new methods. ~ w ~ c.5 ~ CN D b 3
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--1 O Z O ~ Z © I ~ 0 ~ x ~ D C1J -< c~n = c~' D -~~CG822'9
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GENERAL READING RE IEW 1. De Robertis, E.M., Oliver, G. and Wright, V.E. Homeobox genes and the vertebrate body plan. Scientific American, July, pp. 46-52, 1990. 2. Tabin, C. Retinoids, homeoboxes, and growth factors: toward molecular models for limb development. Cell, vol. 66, pp. 199-217, 1991. 3. Reid, L. From gradients to axes, from morphogenesis to differentiation. Cell, vol. 63, pp. 875-882, 1990. 4. Capecchi, M.R. The new mouse genetics: altering the genome by gene targeting. Trends in Genetics, vol. 5, pp. 70-76, 1989. ARTICLES 5. Balling, R., Mutter, G., Gruss, P. and Kessel, M. Cranialfacial abnormalities induced by ectopic expression of the homeobox gene Hox 1.1 in transgenic mice. Cell, vol, 58, pp. 337-347, 1989. 6. Simeone, A., Acampora, D., Nigro, V., Faiella, A., D'Esposito, M., Stornaiuolo, A., Mavilio, F. and Boncinelli, E. Differential regulation by retinoic acid of the homeobox genes of the four HOX loci in human embryonal carcinoma cells. Mechanisms of Development, Vol. 33, pp. 215-228, 1991. co w c.~'j co 0 ~ cn ~ W •
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~ STRUCTURAL ORGANIZATION OF THE MAMMALIAN CHROMOSOME alphoid DNA repeats satellite DNA repeats ! 12 8 B r.= tt;~~+= ~ -Telomere repeats Er; .a& v-v-)r, G+ Bands - Inactive region of chromsome ( ec-mP"$-r' G- Bands - Transcriptionally active regions (loops) - Potential "hot spots" for chromosome re-arrangements - ~ w ~ - ~ ~ ~ ~ ~ .~. D'3 4:
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i MUTAGENIC MECHANISMS IN HUMANS METHODS FOR THEIR DETECTION AND ANALYSIS PRESENTATION OUTLINE • NATURE OF MUTATION IN HUMANS - Source of new mutation - Consequences of mutation - Mechanism responsible for mutation - DNA repair processes CONVENTIONAL METHODS EMPLOYED TO DETECT DNA LESIONS - Core Battery for genetic toxicology - Limitations of the methods NEW METHODS PROPOSED FOR INVESTIGATING GENETIC TOXICITY - Requirements - SSCP/PCR - GGE/PCR - Shuttle vectors and transgenic models D aA
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• David J. Brusick, P.H.D., A.T.S. Scientific Director _Ha.a+et~n Washington, Inc. 04Vr1 nC6- • Dr. Brusick was awarded his doctoral degree in microbial genetics by Illinois State University in 1970, and did postdoctoral research as a National Academy of Sciences research associate at the Food and Drug Administration's Genetic Toxicology Branch. Dr. Brusick is past president of the U.S. Environmental Mutagen Society (1978-79) and is adjunct associate professor of microbiology and genetics at Howard University Medical School and George Washington University, respectively. He is the author of numerous scientific publications, including a textbook, Principles of Genetic Toxicoloev (Second Edition, 1987) and is the editor of In Vitro Toxicology, a journal of cellular and molecular toxicology. Dr. Brusick has served as a member of numerous NAS committees and chaired an NAS/NRC subcommittee on the role of DNA adducts in toxicology testing. He is chairman of the International Commission for the Protection Against Environmental Mutagens and Carcinogens and a member of the Technology Transfer Committee for the Center for Alternative to Animal Testing at John Hopkins University. Dr. Brusick is a fellow of the Academy of Toxicological Sciences and is currently the scientific director for Hazleton Washington. His interests include basic and applied research in mutagenic and carcinogenic mechanisms and the application of biotechnology techniques to safety testing method development. 00 t1J C!J ~ ~ DP~ l
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! DNA DAMAGE ASSOCIATIONS GERM CELL Genetic Diseases (Direct/Indirect) Congenital Anomalies ~ ~ ~_p,, ~ 40 ~ ?1;Ai SOMATIC CELL Cancer Initiation Atherosclerosis Fetal Anomalies co w w co u\ r".) wi3 4
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0 GENETIC MECHANISMS IN HUMANS Numerical Chromosome Alterations . Aneuploidy . Polyploidy Structural Chromosome Alterations . Deletions . Translocations Base Pair Substitutions . Structural Gene . Regulatory Gene Homozygosis Addition / Deletion Mutations Methylation of Cytosine - .• Gene Duplication - ~~ ~~
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GENOME ORGANIZATION IN HUMANS GENOME Human genome ~ 3 x 109 bp (-3000 Mbp) Average chromosome _ 1 x i 08 bp (-100 Mbp) Average chromosome band _ 3 x 106 bp (-3 Mbp) GENES Very small (neuropeptides) _ 0.1 kbp Intermediate (f3 globin) _ 1.5 kbp Very large (dystrophin) _ 2,300 kbp • D35
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DNA LESIONS IN MAMMALIAN CELLS Occurance Is Not A Rare Phenomenon Exogenous Exposures • Radiation - radon gas • Pollutants - hydrocarbons • Foods - pyrolized amino acids, aflatoxin • Drugs - chemotherapy for cancer, psoriasis • Medical Intervention - example: sickle cell disease 1972 14 average survival age 1990 42 average survival age Endogenous Exposures / Factors . Oxidative Processes - lipid peroxidation - peroxisome proliferation . Metabolic Intermediates - formaldehyde and other aldehydes - nitrates-+nitrosamines . Hormones • i aB i3
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EXCISION REPAIR: METHODS LEADING TO SHORT AND LONG PATCH REPAIR OF DNA NUCLEOTIDE BASE EXCISION REPAIR EXCISION REPAIR Helix distorting defect Base defect ~ 000000 00000000 Defective step in XP & rodent ERCC complementa- tion groups Base removal Recognition of bY glycosylase damage & incision, requiring ~ ~ -10-20 proteins -0=" V V V 00000000 I Incision by AP + endonuclease ZT= -770= 0 0000000 Excision & Excision & polymerization polymerivation p V r6W V V V~ v V v v 00000000 00000000 I Ligation 00001110000 LLong patch repair I Ligation 0090000 00000000 Short patch repair Long-patch (100-300 nucleotides) initiated by bulky adducts which distort the helix. Short-patch (single base to a few bases) initiated by glycosylase enzymes which recognize modified bases (e.g., hydroxymethyl uracil) and replace them using apurinic/apyrimidinic (AP) nucleases. Following base removal, excision of one to a few bases occurs followed by ligation. ! DB `i
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INDUCED MUTATION AND DNA REPAIR 0 . MUTATION OCCURS AS THE RESULT OF ALTERATION TO THE NUCLEOTIDE SEQUENCE WITHING A GENE FOLLOWING CHEMICAL OR RADIATION EXPOSURE. C A T i T C A; C C T G T A C C Ai NORMAL G T AiA G TiG G A~C A TIG G T~ i r 1 i t 1 ~ ~ r I 1~ i 1 t F 1 i T~ ~ 1 Y 1 I I 1 C A TIG C AiC C TiG T AjC C Al G T AIC G TtG G AiC A TiG G Ti t t trl t 1, ta/ t/,t SUBSTITUTE ONE BASE PAIR C T G; T A 616 A G; DELETE ONE G T A1 G T GiG A CiA T GjG T Cl BASEPAIR ~ i i 1 1+ 1 r ~ C A T jG T C i A C C t T G TI A C Ci INSERT ONE G T A C A GiT G G A C A T G Gj 1 BASE PAIR - 1 t ~ r I I{J I / y r F i 1 1 A\ C A Tj IC A 6 Dy7 0
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. EXAMPLES OF HUMAN GENETIC DAMAGE ASSOCIATED WITH THE THREE MAJOR CLASSES OF MUTATION 0 Mutation Type Examples of Examples of Inherited Effects Somatic Effects Single base changes Sickle cell disease, Epithelial cancers, Phenylketonuria activation of ras oncogenes Small deletions Haemophilias, Lymphomas, and/or translocations Duchenne muscular leukaemias, dystrophy enhanced activation of myc, abl : oncogenes Whole chromosome Down syndrome Loss of tumour losses or gains Turner's syndrome suppressor genes, retinoblastoma, Wllms' tumour, breast cancer .Dg j0
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i ...4.,.,. Y The Human Genome a9 I-OB22$ II 17 2 12 Aawixx Yu.....1. .MLA.Ma .n., ,_,. ~ ~ ~,w .,....... 18 ""..x. o w.."..., m.. n...rnn...n. .~...x..«.,.««.... J ~ ' h..nF WY.w y y ~ .Y(ryn4wM.. LU. Canryutpanyulul. `~' , ~AJ.m.MM - - V y In w.(«.~q ~". aR r (... „.. ..~w,n.. ~. i 13 19 3 8 :,~.~..n...,.... Atw./.M..L.p. +m.« ».-.. .....w.....4-- ~4w..ae..x. w..w..... ...«4xne."~"..a fu.... ti..4..,. L~.up..M.n(w..w 14 20 Cwx.Ml.... nnu. (.....n.M,. w.+......hM L~.~.~4w•xfl ... :::.,,~ 4 9 ~.~j .....~nm..~.~ IV 21 0 S 10 16 22 A"uu. A uM4n. aa.... ~...,.. G.u4 n.ela.
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0 A protein molecule called p53 plays several roles to protect cells and organisnxs from genetic damage but, as the lower panel illustrates, when p53 itseljis damaged, cells can tura cancerous. WITH NORMAL P53 If damage is repaired, p53 allows cell division to proceed. r QDNA damaged by © p53 halts cell division and heat, radiation or stimulates damage-repair enzymes If damage is too extensive, p53 chemical carcinogen. to rebuild damaged region. commands cefl to commit suicide. © DNA damaged by © Cell is free to divide heat, radiation or without DNA damage chemical carcinogen. being repaired. ® Cells with DNA damage continue to divide. More damage may accumulate, turning cell . cancerous. ~ C~J W ~ .. ~ D 3 L-'I
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COMPARATIVE REPAIR IN MAMMALIAN CELLS Organ Level . Proliferating > Non-proliferating Chromatin Level . G-bands (Loop DNA) Individual Gene Level . Transcribed > Non-transcribed Type of Damage Level . Bulky > Methyl / Ethyl groups 0 9 Da it7
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Translocation Malignancy onc or activated/ rearranged gene t(2;8)(p11-13;q24) Burkitts lymphoma c-myc (8q24) t(8;14)(q24;q32) ALL-L3 IgH, IgK,1gl t(8;22)(q24;qll) t(8;21)(q22;q22) ANLL-M2 ets-2 (21q22) t(9;22)(q34;q11) CML c-abl (9q34) (also ALL) bcr (22q11) t(11;14)(q13;q32) B cell leuk/iymph. bcl-1 (11q13) t(14;18)(q32;q21) B cell foll.lymph. bcl-2 (18q21) t(15;17)(q24;q21) ANLL-M3 c-fes(15q23) c-erb A2 (17q21) t(14;14)(q.11;q32) T-CLL TCRa (14q11) Tcl-1 (14q32) t(8;14)(q24;qll) T-ALL TCRa (14q11) c-myc (8q24) t(6;9)(p21;q34) ANLL c-pim (6p21) • • I-)s it
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I. ENDOGENOUS EXPOSURES 4 x 103 - 5 x 103 DNA Lesions / Hour / Cell 1014 Cells / Person 4 - 5 x 10" Lesions / Person / Hour Estimated repair rates give a reasonable excess in capacity (2-4 times) II. EXOGENOUS EXPOSURES Generally unknown for most agents, but estimates range from 5 - 500 lesions per cell / exposure 5 x 1016 Lesions / Person / Exposure Consequently, the number of exposures and type of lesions produced may be critical ~ v cN ! 17ii i5
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• APPROXIMATE RATES OF DNA DAMAGE AND REPAIR IN HUMAN CELLS AT BODY TEMPERATURE ype of Damage Estimated Occurrences of Damage per Hour per Cell° Estimated Maximal Repair Rate,. Base Pairs per Hour per Cella Endogenous Depurination 1,000 h Depyrimidination 55 b Cytosine deamination 15 b Single-strand breaks 5,000 2 x 105 N7-methylguanine 3,500 Not reported 06-methylguanine 130 104 Oxidation products 120 105 Exogenous Background ionizing radiation Single-strand breaks 0-4 x 105 Oxidation damage 10'4-10-3 105 Ultraviolet irradiation of skin (noon Texas sunlight) Primidine dimers 5 x 10° 5 x 10° 00 a Might be higher or lower by a factor of 2 (Setlow, 1983) , w b Not reported, but the rates are at least 104 ob `j t~j D 9 4
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• DEVELOPMENT OF NEW METHODS FOR GENETIC TESTING 0 I. Broader array of lesions detected II. Nucleotide sequencing (fingerprint) III. In vivo rather than In vitro co U4 tA i 05 Cn ~ Cn iJ B -2A
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ORGANIZATION OF EUKARYOTIC GENES intervening sequences (noncoding introns) i-ONA (chromatin) poly (A) coding sequences (exons) promoter addition site gene pre-mRNA (3') (5`) mature mRNA r I transcription (RNA polymerase) RNA processing (splicing, capping polyadenylation) (31) AAAA W) -•-- 3' nontranslated leader coding 5' nontranslated region region sequence sequence export into cytoplasm I :........................................................................................ translation (ribosome) ~~ NH2 nascent '*ip^^NHy polypeptide polyribosome (3' ) AAA " FT f ribosome polypeptide processing (phosphorylation etc., secondary proteolysis) Lb-final polypeptide Prokaryotic organisms do not bave introns 0 ! j) g ~5
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GENOTOXICITY CORE BATTERY 1. Ames Test 2. In Vivo Cytogenetics 3. Mammalian Cell Mutation Assay (FDA and EC yes; others, no) 4. In Vitro Chromosome Aberrations (MOHW, yes) FDA Proposal: Do mouse lymphoma TK mutation assay or Do CHO/HGRPT mutation assay plus in vitro chromosome aberrations D5 N • co W c.w co ` 0 _ ~ ~ V
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NEW METHODS FOR MUTATION IDENTIFICATION IN VIVO I. Single-strand conformation polymorphism / PCR (SSCP/PCR) • In vivo / any organ • Non-Selective • Detects one-several base pair changes • Sequence mutants II. Gradient Gel Electrophoresis / PCR ~ (GGE/PCR) • In vivo / spleen T-cells • Selective (resistance to 6-Thioguanine) • Detects one-several base pair changes • Sequence mutants III. Transgenic Systems • In vivo / any organ • Selective • Detects one-hundreds of base pair changes • Special mouse strains • Sequence mutants ~, 04 Cd C~) ~ ~ C11 ~ 3g9 C z4" ) N) L) aat
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0 LIMITATIONS OF CORE BATTERY AMES TEST - Prokaryotic DNA, differing gene organization and regulation - Cannot assess site or nucleotide change related to mutation - Reverse mutation involves restricted target site - Higher probability of locus specificity due to restricted target site - Cannot detect recombinational events CYTOGENETICS IN VITRO - Cannot detect small rearrangements or deletions - Susceptible to secondary effects due to in vitro exposures - CHO cells have less 06-demethylose repair activity than primary cells IN VIVO 0 Requires toxic dose levels to achieve acceptable exposures - Cannot detect small rearrangements or deletions Aneuploidy or polyloidy not reliably detected CHO HGPRT - Hemizygous and cannot detect some mutation mechanisms MOUSE LPMPHOMA - Susceptible to non-genotoxic effects *IN VITRO EXPOSURES INCREASE RISK OF FALSE POSITIVE RESPONSES *CELL LINES USED IN VITRO RATHER THAN PRIMARY CELLS *NO TOXICOKINETIC DATA *DIFFERING REPAIR IS POSSIBLE *RELEVANCE TO CELL TRANSFORMATION Co csj W e-s ~-: D 8 -ao
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. TECHNOLOGY TRANSFER Shuttle vector technology created the opportunity to develop a true In Vivo Mutation Model with high resolution and broad tissue accessability including germinal tissues. 0 D E331
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NUCLEOTIDE SEQUENCE The ability to sequence mutants might provide information that will result in mutagen "signatures" or "fingerprints" UY PI-•GC 0 . AT-•CG Controls AJ-CG Efhylene oxitle-exposed Smokers Data are from hprt gene of human T-cell clones derived from blood i DB -X -1
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PROBLEMS ENCOUNTERED WITH CONVENTIONAL TESTING METHODS I. All lesions found in humans not detected by assays • Aneuploidy • DNA Methylation • Transposons II. Mechanisms producing similar genetic effects may differ • Repeat sequences • Homozygosity • Splice site mutations (not possible in prokaryotes) • III. Locus specificity may affect detection of some chemicals • Hot spots (ras, p53 codons) ps3 'e", • Viral insertion points IV. Repair mechanisms may differ ~-~y~l ~~~~y~~ • 06-demethylase i. /" _ A~ - 2tG V • Coding sequences vs. noncoding ~ V. In vitro -i In vivo extrapolation • Dose setting at toxic concentrations not achievable in vivo • Cell cycle stringency • Phase I and II metabolism absent in vitro 0 i) 13 1g'
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GENES AND CANCER Genetic predisposition to cancer is controlled by genes (oncogenes/tumor suppressor genes) which inherited as heterozygotes. The normal alle/e keeps cell proliferation in check but if genetic alterations occur to make the alleles homozygous, cell proliferation may ensue. An example using the single gene retinoblastoma tumor is shown below with a list of the types of genetic changes that might lead to homozygosity. _ Mechanisms that produce homozygosity or hemizygosity at the retinoblas- toma locus f rb- Chromosom a I nondisjunction and loss Chromosomal nondisjunction and reduplication i rb- Mitotic recombination Tr6- Translocation and deletion Translocation and gene inactivation 1 Q rb - Point mutation An individual has inherited only one active allele at the retinoblastoma locus. Inactivation at the second allele in a somatic cell, leading to loss of replication control, may occur by any of the illustrated mechanisms. • M 0 b(3-~ I
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0 COMMERCIALIZATION Although several potential mutation models were developed, two have reached commercialization including patent protection. Animal Recovery Source Target Locus Reagents Patent Stratagene lad Stratagene US Corning HRP lacZ Ingeny US/EC co b13 3 +
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0 EXAMPLE OF RESULTS FROM SSCP/PCR ANALYSIS OF HUMAN DNA ~.`~C'p~ G~\uG~~VGy~,VG5wG6wG'l~.`~Gg9 CNCNCNCNCMCNCNCN 0 SSCP analysis of fragments of 139 base pairs carrying the sequence of exon 7 of the p53 gene. Genomic DNAs from primary non-small cell carcinomas of the lung (indicated as C), a metastatic tumor (indicated as M) and non-cancerous tissues of the same patient (indicated as N) were analyzed. Electrophoresis was performed in 5% polyacrylamide gel with 10% glycerol at 40 W at room temperature. CN trl t7:J ~ ~ C?) CN Ja _Q Y
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SINGLE-STRAND CONFORMATION POLYMORPHISM ANALYSIS WITH PCR ~ 1 Select Genomic DNA DNA Denature The Double Stranded Product pc R 1 Apply Single Stranded DNA to Non-Denaturing Polyacrylamide Gel 1 Mobility Shifts Due To Single Base Change Can Be Detected 1 Sequence DNA ! b 9 -q7
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Pesticides: Metabolism and Mechanisms of Toxicity 1. Pesticides - Chemical and Use Classes • On W &J CO . CD N-) n w..t
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• Ernest Hodgson 0 Dr Ernest Hodgson is William Neal Reynolds Professor of Toxicology at North Carolina State University. He received the B. Sc. degree with honors from the University of Durham, England and the Ph. D. degree from Oregon State University. He has been on the faculty at NCSU since 1961. Research interests include the role of oxidative enzymes in the metabolism of pesticides in target and non- target species, resistance to pesticides and the biochemistry and molecular biology of the flavin-containing monooxygenase and cytochrome P450. Dr Hodgson is editor of the Journal of Biochemical and Molecular Toxicology and of Reviews in Toxicology. He is also co-editor and part-author of two toxicology textbooks (A Textbook of Modern Toxicology and An Introduction to Biochemical Toxicology). He has received a number of awards, including both the Education and the Merit awards from the Society of Toxicology and the Burdick and Jackson Award from the American Chemical Society, and has served on grant review panels for NIH, EPA, NASA, DOD and others.
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GRADIENT GEL ELECTROPHORESIS AND PCR Mutants Wildtype Clone Die I Amplify HRPT DNA PCR Spleen T-cells +][L-2 (107) Denaturing Mis-Match Gel: Mis-matched -AT-- bases result in - A TA-- DNA opening and retardation Sequence mutant DNA for identification of altered codon(s) is • • Da~Y
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~ APPLICATION OF THE LACTOSE OPERON TO MUTATION DETECTION lacl ~J lad monomeric subunit Transaiption & Translation IacO Tetramer binds the lac operator to prevent A lacl ___-w A NA Polymerase 8 0 a LacZ protein from phage laci ap RNA Polytnerese Carbozy-terminus ot LacZ 6nm badaia ctromosome Functionaf Q -*actosdaseprolefn 0 aco alac Z (y, trensaiption of IacZ Transcription Mutant Iacl repressw does not prevem transaiption ot a lacZ / b B 3,;1
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• EXPECTATIONS • Provide confirmation for In Vitro gene mutation assay results. • Improve "toxicological" perspective for mutation assessments (e.g., metabolism, phase I and II detoxifications, route of exposure). • Ability to couple biomarkers with biological effects (e.g., target tissue specificity, germ cell susceptibility). • Ability to study the role of "secondary" effects on mutation induction and expression (e.g., promoters, cell proliferation, antioxidants and antimutagens). • Direct detection of germ cell mutagens without need for F 1 analysis. 0 UB35
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0 DISTRIBUTION OF CLASSES OF BASE PAIR CHANGES INDUCED INTHE MOUSE HRPT GENE BY ETHYLENE OXIDE MUTATIONS INDUCED IN VIVO IN HPRT EXON 3 OF ETO-TREATED B6C3F1 MICE Base location, mutation observed, and animal from which mutations were isolated are given. Base 135 is defined as the first base in exon 3. 12-day-old mice (n = 3 or 4/group) were given single i.p. injections of 120 mg ETO/kg daily for 5 days (mouse 15, 16, 26 and 27) or 100 mg ETO/kg every other day to achieve cumulative doses of 600 (mouse 7 and 9) and 900 mg/kg (mouse I and 2). Base Mutation Animal designation 145 GC~AT 2 151 GC->AT 15 196 AT -t GC 26 0 196 AT- GC 27 203 AT -CG 1 203 AT-CG 1 203 AT-GC 1 207-212 + G 1 207-212 + G 2 207-212 + G 7 207-212 + G 26 a 222 GC->TA 16 229 GC -> TA 9 271 AT-+TA 2 307 AT-TA 2 a 4 of 20 hprr- mutations from mouse 26 were +G frameshifis and may represent a mutation amplified in vivo by clonal expansion. 01330
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Microinjection Method ! . j) 9 3.3
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. RESPONSES FOR CHEMICALS TESTED IN THE MOUSE SPECIFIC LOCUS ASSAY AND TRANSGENIC MUTATION MODELS Chemical MSLa TGb Strain Acrylamide /+ + lacZ Chlorambucil -/+ + lacZ Diethylnitrosamine -/(-) + lacZ Ethyl methanesulfonate -/+ + lacZ Mitomycin C +/(+) + lacZ Procarbazine +/+ + lacZ Benzo[a]pyrene -/(-) + lad Cyclophosphamide /+ + lad + Ethyl nitrosourea +/+ + lacl Ethylene oxide -/+ + lad Methyl methanesulfonate lad Methyl nitrosourea +/+ + lad Urethane -l- + lad aMSL: mouse specific locus test results. Data are from Bentley et al. [1994]. Responses in spermatogonial/postspermatogonial stages are reported, respectively. + or -, data are conclusively positive or negative; (+) or (-), mutants or no mutants were observed, respectively, but results are inconclusive [see Bentley et al., 1994]. bTG: transgenic mouse mutation assay results. 0o w ~ Adapted from: Gorelick, N.J., Environ. Mo1. Mutagen., 25:218-230, 1995. co ~ ~ pg38
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• PESTICIDES: METABOLISM AND MECHANISMS OF TOXICITY.
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0 TOXICOLOGICAL PERSPECTIVE Mutation Chemical Tissue Response In Vitro Urethane Lung + Benzene Spleen + Acrylamide Bone Marrow + 0 1-3 Butadiene Lung + + pB 3 G;
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AGRICULTURAL CHEMICALS FERTILIZERS PLANT GROWTH REGULATORS ANIMAL GROWTH REGULATORS PESTICIDES ALGICIDES FUNGICIDES HERBICIDES NEMATOCIDES MOLLUSCICIDES INSECTICIDES SYNERGISTS ACARICIDES RODENTICIDES 0 •
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FUTURE • Transgenic model(s), which are faster, cheaper and a more robust assay, can/will replace current tests (e.g., TIC, HPRT, Specific Locus, SLRL). • Transgenic model(s) can/will simplify rodent male germ cell analysis. • Transgenic model(s) can/will define the mechanisms of antimutagens and co-mutagens and proliferating agents. • New transgenic model(s) based on plasmid recovery (lacZ) can/will improve the efficiency of transgenics by increasing the range of mutations detected (e.g., deletions). • 0 0 D a39
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BIOMARKER / BIOLOGICAL EFFECTS COMPARISON Characteristic Big Blue® (lacI) Sensitivity Specificity + Predictivity - Predictivity Overall concordance Overall concordance for the Salmonella/microsome test for this set of chemicals MutaMouseTM (1acZ) Sensitivity Specificity + Predictivity - Predictivity Overall concordance Overall concordance for the Salmonella/microsome test for this set of chemicals Response 67% (12/18) 100% (3/3) 100% (12/12) 33% (3/9) 71% (15/21) 67% (14/21) 83% (10/12) From: Gorelick, N.J., Environ. Mol. Mutagen., 25:218-230, 1995. 0 i 0 b S 37
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e2 Aff.nnt N.rv• Eff.r.nt N.rv• Figure 22-5. A simple, intact refkz arc involving a peripheral, af- ferent (sensory) neuron, interneurons in the CNS, and a peripheral, efferent (motor) nearon that innervates a muscle. From: Ecobichon, D. J. 1996. • r i
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! PERCEIVED RISK 1. PESTICIDES ARE SYNTHETIC CHEMICALS THAT ARE RELEASED PURPOSELY INTO THE ENVIRONMENT. 2. PESTICIDES ARE DESIGNED TO BE TOXIC TO SOME FORM OF LIFE AND OFTEN HAVE THE POTENTIAL TO CAUSE ADVERSE EFFECTS IN NON- TARGET SPECIES, INCLUDING HUMANS. c 3. HUMANS ARE UNAVOIDABLY EXPOSED TO PESTICIDES, EITHER OCCUPATIONALLY OR INVOLUNTARILY THROUGH PESTICIDE RESIDUES PRESENT IN AIR, FOOD AND/OR WATER. ~
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9 0 0 Table 1.2 Classficationn of Pesticides, with Examples Class Algicide Fungicide Principal Chemical Type Organotin Dicarboximide Chlorinated aromatic Dithiocarbamate Mercurial Herbicide Nernatocide Molluscide Amides, Acetamides Bipyridyl Carbamates, Thiccarbamates Phenoxy Dinitrophencl Dinitroaniiine Substituted urea Triazine Halogenated alkane Chlorinated hydrocarbon Example, Common Name 8restar Captan Pentachlorophenoi Maneb Phenyimercudc acetate Propanil Paraquat Barban 2,4-0 DNOC Trifluralin Monuron Atrazine Ethylene dibromide (EDB) 8ayluscide 2
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Table 4.1 Enzymes important in catalyzing metaboiic activation reactions. Type of Reaction Enzyme Oxidation Cytochrome P450s Prostagiandin synthetase (PGS) " Flavin-containing monooxygenases (FMO) Alcohol and aldehyde dehydrogenases Conjugation Glutathione transferases Sulfotransferases Glucuronidases De-conjugation Cysteine S-conjugate f3-lyase Hydrolysis and reduction Gut microflora: hydrolases, reductases From: Hodgson, E. and Levi, P. E. A Text- book of Modern Toxicology, 2nd Edn. 1997 . i
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0 • Acarides Rodenticides Organosulfur compounds Fortnamidine Dinitrophenols DDT anaiogs Anticoaguiants Botanicals Alkaloids Glycosides Fluorides Inorganics Thioureas From: Hodgson, E. and Levi, P. E. A Text- book of Modern Toxicolog_y, 2nd Edn. 1997 Ovex Chlordimefonn Dinex Chforobenzflate Warfadn Slrychine sulfate Sciilaren A and H Fluoroacetate Thallium sulfate ANJ_U 10
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0 Comparison of the Characteristics of Acute versus Chronic Toxicity To be distributed on site co tnt . OIJ CX+ h~9 t ~
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• Exposure ~ Absorption at Portals of Entry ~ ~ Distribution to Body 1 Metabolism to More Toxic Metabolites ~ ~ Metabolism to Conjugation Products Metabolism to Less Toxic Metabolites Distribution ~ ~ • Interaction with Macromolecules (Proteins, DNA, RNA, Receptors, etc.) i ~ Toxic Effects (Genetic, Carcinogenic, Reproductive, Immunotoxic, etc.) From: Hodgson, E. and Levi, P. E. A Text- book of Modern Toxicology, 2nd Edn. 1997 -0- Excretion A Turnover and Repair t:d w ' CYq ~ c.~ tv CD CO
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• Organochlorines pyrethroids ~ Eniymes I(( ..unal raameran.a Iana ( Na* , K*, Ca -. CI' I Eniymes ~ Naaraven.miu.n Organophosphoros and Carhamato Este rnf~.4e~'Iy~/J~/n ~ Figarell-1. Potentiaf sites of action of cfasses of insecticides on the (~ '.mn and the terminal portions of the nerve. ~ ~ ~ From: Ecobichon, D. J. 1996. ~ ~ C:)
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l-3 ! Insectidide Chlorinated hydrocarbons DOT analogs DOT Chlorinated alicyciic 6HC Cyclodiene Aldrin Chlorinated terpenes Toxaphene Organophosphate Parathion Carbarnate Carbaryl Thiocyanate Lethane Dinitrophenols DNOC Fluroacetates Nissol 9otanicals Nicotinoids Nico6ne-C-- Rotenoids Rotenone Pyrethroids Pyrethrin Synthetic pyrethroids Fenvaierate Juvenile hormone analogs Methoprene Growth regulators Dimi(in Inorganics Arsenicals Lead arsenate Fluorides Sodium floride Microbials Thuricide, Avermectin Insecticide synergists Methylenedioxyphenyl Piperonyl butoxide Dioarboximides MGK-264 01 10
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Z-rL) ~ Acute versus Chronic Toxicity Factors Acute Toxicity Chronic Toxicity Exposure Single, repeated or Repeated and prolonged frequency continuous Duration 24 Hours or less At least one-half of the life-span; less in humans Pharmaco- Blood level high, Gradual build up of blood kinetics bioavailable for a level; bioavailability short time prolonged Responses Tmmediate„or in a Delayed, prolonged short time (days). (months, years)_ Target organs Usually few Diversified Mode of action Usually different in acute and chronic toxicity from the same compound. . i
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0 10 ! TABLE 9.4. EXAMPtES OF TOXIC EFFECTS FOLLOWING EXPGSNRE TO NEllROTDXICANTS Motor effects Convulsions Weakness Tremor, nvitcning Lack of coordination Pararysis Rellex abnormalities Mood and personality effects Sleep disturbances Excitability Depression Imtabillty Restlessness Delirium, hallucinaNons Nervousness,tension CagnitJve effects Memory problems Conlusion Speecnimpartmen[ Learning rmpairment Sensory effects Vision disorders Audilory disorders Pain disorders General eneas Narcosrs Fatigue Loss of appeute Stupor From: Hodgson, E. and Levi, P. E. A Text- book of Modern Toxicology, 2nd Edn. 1997
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Table 11.3 A Summary of Tests for Toxicity 1. Chemical and physical properties For the compound in question, probable contaminants from synthesis as well as intermediates and waste products from synthetic processes. 2. Exposure and environmental fate A. Degradation studies - hydrolysis, photodegradion, etc. B. Degradation in soil, water, etc, under various conditions C. Mobility and dissipation in soil, water and air 0. Accumulation in plants, aquatic animals, wild terrestrial animals, food plants and animals, etc. 3. In vivo tests A. Acute L050 and/or LC50 - oral, dermal, or inhaled Eye irritation Dermal irritation Dermal sensitization B. Subchronic 90-day feeding 30 to 90.day dermal or inhalation exposure C. Chronic Chronic feeding (including oncogenicity tests) Teratogenicity Reproduction (more than one generation) D. Special tests Neurotoxicity (delayed neuropathy) Potentiation Metabolism Pharmacodynamics Behavioral 4. !n vitro tests A. Mutagenicity- prokaryote (Ames test) B. Mutagenicity - eukaryote (Drosophilia, mouse, etc) C. Chromosome aberration (Drosophilia, sister chromatid exchange, etc) 5. Effects on wildlife Selected species of wild mammals, birds, fish, and invertebrates: Acute toxicity, accumulation, and reproduction in laboratory simulated field conditions or actual field conditions. From: Hodgson, E. and Levi, P. E. A Text- book of Modern Toxicology, 2nd Edn. 1997 V1 0 Is
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)-g ! THE EFFECT OF PESTICIDES ON HUMAN HEALTH 0 1. PESTICIDES HAVE THE POTENTIAL TO CAUSE A NUMBER OF ADVERSE CHRONIC HEALTH EFFECTS IN NON-TARGET SPECIES, INCLUDING H(]MANS. 2. LEVELS OF HUMAN EXPOSURE MAY BE SIGNIFICANT FOR CERTAIN OCCUPATIONAL GROUPS (FORMULATORS, APPLICATORS, FARM WORKERS, ETC.) BUT ARE TYPICALLY VERY LOW FOR MEMBERS OF THE GENERAL POPULATION. 3. STUDIES WITH LABORATORY ANIMALS AND IN VITRO MODEL SYSTEMS HAVE ESTABLISHED THAT PESTICIDES HAVE THE ABILITY TO UNDERGO A LARGE NUMBER OF MECHANISTICALLY DISTINCT INTERACTIONS WITH A WIDE NUMBER OF BIOLOGICAL TARGETS. 4. THERE IS SOME CONCERN THAT CHRONIC ADVERSE HEALTH EFFECTS IN HUMANS MIGHT RESULT FROM EXCESSIVE (HIGH LEVEL, FREQUENT, LONG-TERM) PESTICIDE EXPOSURES THAT COULD OCCUR IN AN OCCUPATIONAL SETTING. HOWEVER, ALTHOUGH INSUFFICIENT DATA EXIST, THERE IS CURRENTLY LITTLE EVIDENCE TO SUGGEST THAT SUCH EFFECTS ARE LIKELY TO RESULT FROM THE TRACES OF PESTICIDE RESIDUES TO WHICH THE GENERAL POPULATION IS CURRENTLY EXPOSED. ~ 5. THERE IS NO EVIDENCE THAT CURRENT LEVELS OF PESTICIDE RESIDUES IN FOOD ARE CAUSING ANY ACUTE OR CHRONIC ADVERSE HEALTH EFFECTS IN THE US POPULATION. WORKING GROUP TASK FORCE ON ENVIRONMENTAL CANCER AND HEART AND LUNG DISEASE. 1990. co W W ~ co ~ h7 . .'W.
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CH30 I DC7f 113 OCH3 '=CI t Structure Name LD50 Go Methoxychlor 5000 Mirex 600 From: Hodgson, E. and Levi, P. E. A Text- book of Modern Toxicology, 2nd Edn. 1997 J l i !
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0 0 PCR (in vitro DNA amplification) Described as being to genes what Gutenberg's printing press was to the written word, PCR'r,an~ampl'rfy a desired DNA sequence of any origin (virus, bacteria,-piant, or human) hundreds of millions of.times in a matter of hours, a task that would have required several days with recombinant technology. PCRJs espe- cially valuable because the reaction is highly specific, easily automated, and capable of amplifying minute amounts of sample. For these reasons, PCR has also had a major impact on clinical medicine, genetic disease diagnostics, forensic science, and evolutioriary biology. PCR is a process basea on a specialized potymerase enzyme, which can synthesize a complementary strand to a given DNA strand in &mixture'bontaining!the 4 DNA bases and 2 DNA fragments. (primers, each ~,: about 20 bases long) flanking;the~target seqtierice.-The mixture is heated to separate thestrandstof double- stranded DNA containing the tatget sequence and then cooled to allow (1) the primers toaind and;bind to their complementary sequences on the separated strands and (2) the polymerase to extend the pdmers into new complementary strands. Repeated heating and cooling cycles multiply the target DNA-:ezponentialty, . since each new double strandseparates to become1wo templates for further synthesis. In aboutI hour, 20 PCR cycles can amplify the target by a millionfold. Reaction mixture contains target . DNA sequence to be amplified, two primers P1, PP), atM heat-stable eqpolymerase Readion mixture is heatetl DNA Amplification Using PCR TARGET DNA lI11.11JJJJtl1111JJJ1J111 ~ Pt~ • Taq PZ ip95°Ctodenatureterget DNA. SubsequeMOOONngmers M _ ~m ii~~r(( j(((( j 1111111t1lllllsll~ ~ to 3TC albws pri ~~'~~'~t'r~ hybriClze to canplemenqry, sequences in target DNA ur " ,iir.L1~.LI.LLLLLi.LLf,1~.L cJj ~when heatetl to 72'C, TaqpaVymerase etlends oomplementery'' ~ ~~ strands irom primers ~ Q Fust bynthesls cyde resulte 4- ~ ~ LL - 1 tar getDNA sequence f r,,, a %44,, rMTrrT1TnT TP'r DENATURE IIIIIIIIIIIIIIIHIR _ ~ ~ ~ ~ ~~fgli~8~6Y~, Hilillil ~ ~ V Source: DNASdence, see Fig. 11 e DNA IIIIIIillflfr j ~~ 11111 ~ , HYBRIDIZE PRIMERS E%TEND NEw DN J,LLLj.I~-f ulity..,~A STRANDS ( LI1J.Lu IA.LME Second synthesis ryde results in four oopies of target DNA sequence I V 1.J-'l(:
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Structure S n (CzH50)2P-O N02 S N-<CH(CH3)z i N S (CzHsO)zP -S(CH2)rSC2H5 S (CH30hP-SCHCOOCA i CH2COOCZH5 Name LDSO Parathion 3-13 Diazinon 250-285 Disulfoton 2-7 Malathion From: Hodgson, E. and Levi, P. E. A Text- book of Modern Toxicology, 2nd Edn. 1997 ,i.' A • i 00 W U.t CO CD 0 fV v
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AChE Protein (Serine residue) wwwrY.~. CHZ ~ OH (CH~N(CH~OCCH3 Acetylchoiine Chofine + CHz i 0-CCH3 I I 0 Acetyiated Enzyme Hz0 (rapid) 4 - Acetate 0 n (CzH50)zPOC6H,N0Z Paraoxon r^^~M + HOC6H,NO2 C~ p-Nitrophenoi i 0-P(OC2H5~ ,4A) , 0 V Phosphorylated Enzyme FizO (very slow) - diethyl phosphate (Y~i Figure 7.1. SchemaUc illustrating hydrolyyis of ACh antl paratnion by the enzyme (AChE). From: Hodgson, E. and Levi, P. E. A Text- book of Modern Toxicology, 2nd Edn. 1997
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Nicotinoids - Structures, Mode of Action and Metabolism. To be distributed on site 5 ~) C!~I •
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5 -v i Pesticides: Metabolism and Mechanisms of Toxicity 5. New Classes of Pesticides - Metabolism and Mode of Action • CZY W ~ C53 fv ~ fX~
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0 . N. K o0 r ~~ oe ~ e a alimGlln n G2 ~ .I f L~~H~J` CazM9 Z.\ ~ ATPap \', Caz• I #4 #3 Figure 22-7. Proposed sites of action of DDT on (1) reducing po' 00 tasstum transport through pores; (2) inactivating sodium channel CN . closure; (3) inhibiting sodium-potassium and calcium-magnesium ~ ATPaser and (4) calmodulin-ca/cium binding with release of neura• ~ transmitter. CJ From: Ecobichon, D. J. 1996. N) ATPea. #z #3 Na• T K' _` #1
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0 Pesticides: Metabolism and Mechanisms of Toxicity 6. Mechanisms of Toxic Interactions of Pesticides
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0 ~/J~ ~ ~ OR E-O-P~ 0 R' Nl~-IC!N-OH I I +CH3 H i ~OR' ~ 2 PAM • EO-P KR \ Ikt OR 0OR Co W E+ I N. ' N-O-P~ C~ OR 1 CN Ob O CH3 H h~) Figure 22-18. The prulidoxime-caralvzed reactivation of an organophosphate-inhibired molecule of AChE, showing the release of active enzyme and the formation of an oxime•phosphare comp(er. From: Ecobichon, D. J. 1996.
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a. Nitro reduction 0 Nitrabenzene Nitrasobenzene Phenyl- hydroxylamine b. Azo reduction N=N---~\ /}NHz N-N Aniline 0-Aminoazotoluene H H ~I ..' v - rvn2 Hydrazo derivative c. Disulfide reduction S S S n (CZH.)zNCS- SCN(C2H,)Z ~ 2 (CZH.)zNCSH Disulfiram d. Aldehyde reduction CI-{' `~--CHD -I.- Dimethyldithio- carbamic acid CI-~ '~-CH,OH p-C hlorobenzal dehyde o-Chlorohenzyi alcohot e. Sulfoxide reduction 0 Amine products (CzHs)ZPSCHzS-~Ct --I- (CzFk)2PSCHzS-(l `}-CI Carbophenothion sulfoxide Carbophenothion From: Hodgson, E. and Levi, P. E. A Text- book of Modem Toxicology, 2nd Edn. 1997 0
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OH + HCHO NO2 L NOZ p-Nitroanisole HCI 0 C 0 C2H3 Czy50~- C! Chlorfenvinphos Ethylmorphine 3 N-CH3 J N02 p-Nitrophenol r OH O CHZCHO,P C2H50 NH . HCHO From: Hodgson, E. and Levi, P. E. A Text- book of Modern Toxicology, 2nd Edn. 1997 ~ HO'p_ + CH3CHO CzHSO .~ 0 0
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Photoxidation V Hydrolysis S (C2H50)2 -P-O Parathion / 0 - (C2H50)2-P-O--~: '/-NO, Paraoxon N02 p-Nitrophenol + S (CzHsO)z -P-O' Oiethvlohosoho rothioate From: Hodgson, E. and Levi, P. E. A Text- book of Modern Toxicology, 2nd Edn. 1997 r \
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0 N(CH3)2 -)P- (I ~)-N(CH3)z 0 A Dimefhyianiline Nicotine-1'-N-oxide N Dimefhylanifine N-oxide aC S CNH2 '}-CNHZ S Thiobenzarnide Thicbenzamide S-oxide O V q~~w~ S S A CZH-O °SCH2SCZH5 -~ CzHSo;PSCHZSCZH. t ~ z Cz s ~j Phorate Phorate sulfoxide i 0 P-CH~ -~ " ~P-CH3 Diphenyimethyl- ohosohine Diphenylmethyi- phosphine oxide From: Hodgson, E. and Levi, P. E. A Text- book of Modern Toxicology, 2nd Edn. 1997 Cd cn+ W (tb ~ CD t-j U4 ~
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Paraquat O Diquat O/ \O Figure 22-22. The chemical structures of paraquat and diquat, mar- keted as the dichloride and dibromide salts, respectively. From: Ecobichon, D. J. 1996.
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! Effect of a single injection of piperonyl butoxide on cytochrome P450 and microsomal protein in mouse liver. 1.8 1.6 1.4 0 0 U 1.2 ~ 1A 0 E ¢ 0 8 ~ . > .~ a 0.6 0 ~ 1 0.4 12 24 36 48 60 Time (hours) 72 84
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0 I" i r a,„jgble 22-7 %gigns and Symptoms of Anticholinesterase Insecticide Poisoning ~ NERVOUS TISSUE AND RECEPTORS _ AFFECTED SITE AFFECTED ' parasympatheic autonomic (muscarinic receptors) postganglionic nerve fibers parasympathetic and svmpathetic autonomic fibers (nicotinic receptors) Somatic motor nerve fibers (nicotine receptors) Exocrine glands Eyes Gastrointestinal tract Respiratory tract Cardiovascular system Bladder Cardiovascular system y45~'4 t,~S i'~ MANIPES'L4TIONS Increased salivation. lacrimation, perspiration Miosis (pinpoint and nonreactive), ptosis, blurrins of vision, conjunctival injection. "bloody tears" Nausea. vomiting, abdominal tightness, swelling and carmps. diarrhea, tenesmus, fecal incontinence Excessive bronchial secretions, rhinorrhea, wheezing. edema. tightness in chest. bronchospasms. broncho- constriction.cough,bradypnea.dyspnea Bradycardia, decrease in blood pressure Urinary frequency and incontinence Tachycardia. pallor. increase in blood pressure Skeletal muscles Muscle fasciculations (eyelids, fine facial muscles). cramps. diminished tendon reflexes, generalized musc weakness in peripheral and respiratory muscles. paralysis, flaccid or rigid tone Restlessness, generalized motor activity, reaction to acoustic stimuli, tremulousness, emotional lability, ataxia Brain (acetylcholine receptors) Central nervous system Drowsiness, lethargy, fatigue. mental confusion. inabiiir to concentrate, headache, pressure in head. generalize weakness Coma with absence of reflexes, tremors. Chevne-Stokes respiration. dyspnea, convulsions, depression of respiratory centers. cyanosis CO i C~l From: Ecobichon, D. J. 1996. CO 0 fV W 01%
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9 9 0 GABA PYRETHROIDS PYRETHROIDS Figure 22-20. Froposed cellular mechanisms by which pyrcthro esters inretfere with neurona( functiorc From: Ecobichon, D. J. 1996.
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! 0 ~~---CHZ N~ ~NH r Imidacloprid ""02 i CyHl0CIN502 ~ MW= 255.66 ~ \\CHZ N- NH CI CH-IMI CHNOZ C10HlICIN40z MW= 254.67 ' H teon t4r-- H3C~ CHs CHZ N\ /NH CHNO2 Nitenpyram CIIH15CIN40z MW= 270.71 CH3 CHZ N\ /CH3 'xN' CN Acetamiprid CIOH11CIN4 MW= 222.67
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0 0 Z
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OH OH + HCHO ,~ Carbene ~ " 1 Complexes with Fer2 of cytochrome P450 to form metabolite inhibitory complex From: Hodgson, E. and Levi, P. E. A Text- book of Modern Toxicology, 2nd Edn. 1997
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OCHzCOOH CI 2, 4-D ichl orop henoxy- acetic acid'(2,4-D) CI 2,4,5-Trichlorophenoxy- acetic acid (2,4,5-T) 2,3,7,8-Tetrachloro- dihenzodioxin (TCDD) From: Hodgson, E. and Levi, P. E. A Text- book of Modern Toxicology, 2nd Edn. 1997 ~ . ep'T' ~'e `~rl 7 k 0 From: Hodgson, E. and Levi, P. E. A Text- book of Modern Toxicology, 2nd Edn. 1997
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! i Purine Alkylation Rm As I ,`-R~ Rr Ra -Y ~e"~"1 I T Sugar Guanine R, NC,"f,i~ Adenine I' Suga. RI,Ri R, -SNy Ri-SNt R3 - aralkylating agents Rq - esters of aromatic hydrozylaroines Adaptcd from: DipFle, A., in Chemical Carcinogens-Activafion Mechanism, Stmctwal and Electronic Factors and Reacfivity, P. Poliaer and L. Robms (eds), Elsvier, 1988 ~
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0 Effect of Induction of Pesticide-Metabolizing Enzymes on Toxicity. To be distributed on site 40 CU W W fv ~ 40 CYJ
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0 Metabolic Formation of Chemically Reactive Species Kenneth Korzekwa Center for Clinical Pharmacology University of Pittsburgh I* co w w CO ~ CD N CS"i ...a 1
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• ELECTROPHILIC SPECIES K. KORZEKWA w
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0 Phase I Drug Metabolism Pathways I* • Oxidations (NrP"~O) - aliphatic oxidation - aromatic oxidation - olefenic oxidation - N,O, and S dealkylation - oxidation of alcohols and aldehydes - others • Reductions 'Uftw - of aldehydes and ketone reduction - nitro reduction - azo reduction - others • Hydrolysis Reactions - hydrolysis of esters and amides - epoxide hydration Phase IDrug Metabolism Pathways is Glucuronic Acid conjugation Sulfate conjugation • Glutathione conjugation - mercapturic acid formation • Amino acid conjugation • Acetylation • Methylation 3
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i 11
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! Adduct Formation Protein nucleophile Protein-Z Lipophilic Electrophilic `' DNA-Z Xenobiotic Metabolite DNA nucleophile Nucleophilic Amino Acids Cysteine Lysine 4
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• Pyrimidine Alkylation NHp ~- Rx 0 rF_ Rx A `@ F R ` Y 1 „ . i x ~- Ry Cytosine N O F~ RZ I s~r Thymine R, -SNZ Rz-SN, R, - aralkylating agents Ry-esters of aromatic hydroxylamines Adapted flom: Dipple, A., in Chemical Carcinogens-Activa4on Mechanism, Sauctural and ElecVOnic Facrors and Reacdvity, P. Pofitzer and L. Robens (eds), Etsviet, 1988 ! Generation of Apurinic Sites Adapted Bom: Rlein et al., Chem.Res.Toxicol.,4,594,1991, 4 • 6
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• Alkylation of Guanine Ofi- and/or N2- reactivity increases Adapted from: Dipple, A., in Chemical Carcinogens-Activatfon Mechanistq Structural and Electronic Factors and Reactivity, P. Politrer and L. Roberts (eds), Elsvier, 1988 I* Glutathione/ Mercapturic Acid Adducts Nliz Hlliuy i HOOC-~ \CHyCHyCnI / ~cH sH HOOCq/iMNOC Glutathione (GSH) GS-E Glutathione Adduct NHCOCHa Hllu/i \cHSE HOOC Mercaptuiic Acid w iJ14 W Cri ~ C:) CJ'7 ---I 7
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Activation of Benzo[a]pyrene soaxlda 101 Y OH (Olal Epoxide I) Hydrolase P450 OH ! 12
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i 9
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0 Episulfonium Formation from EDB NHz HUr„•,I H00C/ \CHpCHZCOI H + IHz Hlpq..C BrCH2CH2Br NHOOC"I ~CHzCHyCONH Hlm•/ \CH,SH HOOCCHzHNOC HOOCCHzHNOC Hllu./I \CH25CHiCHZBz HOOCCHzHNOC NHz Hllbr.,l HOOC~ ~CHpCHyCONH ~~ Hllbu•I r / -CH ~ 28 ~ • The Cytochrome P450 Enzymes • Oxidative Enzymes involved in the metabolism of endogenous and exogenous compounds - Endogenous substrates • steroids • prostaglandins • others - Foreign compounds • drugs • environmental contaminants • 8
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0 Nitrogen Mustards cl 0 H CI 9 N -N: ~. CI 0
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0 CURRICULUM VITAE Name: Kenneth R. Korzekwa July 24, 1995 Date and Place of Birth: December 25, 1957; Hobbs, New Mexico Citizenshin: United States Education: Dec. 1980 - B.S. (Chemical Engineering) New Mexico State University March 1987 - Ph.D. (Medicinal Chemistry) University of Washington (Thesis: Theoretical and Isotope Effect Studies on Cytochrome P-450 Mediated Aromatic Oxidation.) • Brief Chronology_of Employment: 1980-1981 - Chemical Engineer, Union Carbide Corp., Charleston, W V. 1981-1987- Graduate Research Assistant: Dept. of Medicinal Chemistry, University of Washington, Seattle, WA. 1987 - Staff Fellow, NIH, NHLBI, Laboratory of Chemical Pharmacology 1987-1989 - NIH, Pharmacology Research Associate Training Program (PRAT Fellow) 1989-1990- Senior Staff Fellow, NIH, NHLBI, Laboratory of Chemical Pharmacology 1990-1994- Senior Staff Fellow, NIH, NCI, DCE, Laboratory of Molecular Carcinogenesis 1995-Date- Visiting Associate Professor, Center for Clinical Pharmacology; Director, Drug ;Discovery Program, University of Pittsburgh Editorships: Editorial Review Board, Journal of Biochemical Toxicology. Research Interests: Mechanistic studies in drug and steroid metabolism; The use of theoretical methods and stable isotopes to probe chemical and biological systems. Present Home Address: 6526 Dalzell Place co Pittsburgh, PA 15217 C~l 412-421-9344 04 . ~ ~ KK R
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0 ! co w w co C~ n5 w 13
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• Azoreduction NHr HzNOzS Pronrosil HyNOzS Sulfanilamitle 1.2,4-Tnamino6enene Reductive metabolism of CCL4 • CI~C-CI CI CIa 17
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Moslen Page 11 • Endoplasmic reticulum • 1 Influ calc t Cell d x of ium eath (50 W 06 ~ (n Cj V F43 ccl, Cytochrome P„a CCi,• Lipid-H CCI,H 1 '-Lipid• I/ -02 Lipid _ _ peroxides Fragmentation of fatty acid - - peroxides I Release of - - soluble toxins r Damage to plasma membrane
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Esterases CH, ~ N-C-O CHj OH CH, NH-C CH, ~ HO OH CH, NH-C` CH, CH,~ N-C-O CHy p CH, HO CH, BAMBUTEROL TERBUTALINE • • co C7.1 {T7 (DO d ' n) cr. c~ 18
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. RaI MhM N- and O-Dealkylation r ~- ~. I ~ R-O-'~'- l- Hertieceteia hwnYeml Ncahol cmw^H^*t9 I L ~I I. RiNH + __III Po Tertlary/4rine Certindamine Secavisrypnine Cmbanylmdety • 10
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i 0 co w w co ~ ~ i OI' LYI 15
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! Activation of Cyclophosphamide o~ 0 _N~' HyN1IP l W<N i ~ CI r_n O a HyN-p~n~~ \ / 0 O 0 HvNip ©' \~Q II f\ O
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Cl6 0 oll~oH, NH-CCFL~{ / CH.CHa CH Tr Udocaine Esterases/Amidases HsN-Q J)-C-NHCHzCHzN(CHyCHAz Prncainamltle HzN-j( JJ-C-OCHrCHzN(CHzCHah Pmesine 0 Mechanism of Epoxide Hydrolase 0 H:&Enz CO w w~y~ V~+ CD N-) _ CTN .,~ 19
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• The Cytochrome P450 Enzymes in Chemical Carcinogenesis Kenneth Korzekwa Center for Clinical Pharmacology University of Pittsburgh Medical center • The Cytochrome P450 Enzymes • Oxidative Enzymes involved in the metabolism of endogenous and exogenous compounds - Endogenous substrates • steroids • prostaglandins • others - Foreign compounds • drugs • environmental contaminants t\~ ~
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! 0 ! 21
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r Kinetics of hydrolysis of K-region epoxides of PAHs by MEH. PAHs R,S-epoxide S,R•epoxide Km° Vmaxb (V/K)Rs Km Vmax B[a]P 5.6(0.6)° 2.3(0.1) 0.41(0.02) 2.5(0.1) 1.94(0.02) 3MC 13.6(1.4) 0.66(0.05) 0.048(0.002) 4.7(0.2) 2.65(0.05) CR 3.8(0.4) 2.19(0.09) 0.57(0.03) 16.0(1.3) 1.06(0.07) DB[a,h]A 3.2(0.5) 0.43(0.02) 0.14(0.01) 25.7(5.7) 0.50(0.09) DMBA 29.0(7.9) 0.14(0.03) 0.0049(0.0003) 4.0(0.3) 0.115(0.003) ' Kmis eapressed in µM. b Vmax is expressed in nmoles of dihydrodiol formed per minute permg of membrane protein. ~ standard errors in psrentheses. 0 . 20
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0 Are the P450s Good Enzymes? • Most enzymes are: • Many P450s are: - fast - slow - efficient - inefficient lo ~d - selective - nonselective - bioactivators 0 L"
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References 1. Williams RT, Detoxification Mechanisms. John Wiley and Sons, New York, 1971. 2. White RE and Coon MJ, Oxygen activation by cytochrome P-450. Annu Rev Biochem 49: 315-356, 1980. ! 3. Conney AH, Induction microsomal enzymes by foreign chemicals and carcinogenesis by polycyclic aromatic hydrocarbons. G.H.A. Clowes Memorial Lecture. Cancer Res 42: 4875-4917, 1982. 4. Hanioka N, Korzekwa K and Gonzalez FJ, Sequence requirements for cytochromes P450IIA1 and P4501IA2 catalytic activity: evidence for both specific and non-specific substrate binding interactions through use of chimeric cDNAs and cDNA expression. Protein Eng 3: 571-575, 1990. 5. Van Wauwe JP and Janssen AJ, Is There a Case for P-450 Inhibitors in Cancer Treatment? J Med Chem 32: 2231-2239, 1989. 6. Cook JW, Hewett CL and Hieger I, The Isolation of a Cancer Producing Hydrocarbon From Coal Tar. Parts I, II, and III. J Chem Soc 395-405, 1932. 7. Yang SK, McCourt DW, Leutz JC and Gelboin HV, Benzo[a]pyrene diol epoxides: mechanism of enzymatic formation and optically active intermediates. Science 196: 1199-1201, 1977. 8. Jerina DM, The 1982 Bernard B. brodie Award Lecture. Metabolism of Aromatic hydrocarbons by the cytochrome P-450 system and epoxide bydrolase. Drug Metab Dispos 11: 1-4, 1983. 0 9. Armstrong RN and Lacourciere GM, The catalytic mechanism of microsomal epoxide hydrolase involves an ester intermediate. JAm Chem Soc 115: 10466-10467, 1993. 10. Batt AM, Magdalou J, Vincent-Viry M, Ouzzine M, Fournel-Gigleux S, Galteau MM and Siest G, Drug metabolizing enzymes related to laboratory medicine: cytochromes P-450 and UDP-glucuronosyltransferases. [Review] [111 refs]. Clin ChimActa 226: 171-190, 1994. 11. Mantle TJ, The glutathione S-transferase multigene family: a paradigm for xenobiotic interactions. [Review] [29 refs]. Biochem Soc Trans 23: 423-425, 1995. 12. Nebert DW, McKinnon RA and Puga A, Human drug-metabolizing enzyme polymorphisms: effects on risk of toxicity and cancer. DNA Cell Biol 15: 273-280, 1996. 13. Walter-Sack I and Klotz U, Influence of diet and nutritional status on drug metabolism.. Clin Pharmacokfnet 31: 47-64, 1996. 14. Le HT and Franklin MR, Selective induction of phase II drug metabolizing enzyme activities by quinolines and isoquinolines. Chem Biol Interact 103: 167-178, 1997. 15. Armstrong RN, Structure, catalytic mechanism, and evolution of the glutathione transferases. Chem Res ~ Toxicol 10: 2-18, 1997. 8338027?
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0 0 Cytochrome P450 Catalytic Cycle H20 (RH)Fe+3 (RH) Fo+2 0-O ~ (RH) Fe+3 0-0" lµ~~ 00 W (J^! Co CD ~ ~ ``0
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w w cN 00 0 N ~ CIN
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! Some of the P450s appear to have evolved specifically for the metabolism of xenobiotics • This task is accomplished by: - A superfamily of enzymes - Broad and overlapping substrate specificity - A very reactive oxygenating species • The Cytochrome P450 Enzymes • 12 families in mammals • Metabolize endogenous and exogenous compounds • Xenobiotic metabolizing P450s - CYP1 (2 subfamilies) - CYP2 (7 subfamilies) - CYP3 (2 subfamilies) 0
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• The Cytochrome P450s are Reductases • The catalytic steps for the xenobiotic metabolizing P450s are probably associated with oxygen activation zr+zN+ ~ i 1~ ~ + 02 ~ ~ Fe ~ ~ H40 SR • The active oxygenating species is very reactive • can metabolize most functional groups • e.g. hydrogen abstraction from alkanes • addition to unsaturated functionalities r 0
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• Hydrogen Peroxide Release • Hydrogen peroxide release may result from water molecules in the active site - only substrates that can displace water from the active site can be efficiently metabolized • This decoupling pathway may prevent the generation of the active oxygen in the absence of a substrate. 0 Excess Water Formation A-,Q~-Z-~ Yw"~t le- 3,-d e " • Reduction of the active oxygen to water: - is in competition with substrate oxidation - is initiated by the introduction of the 3rd electron •'rhis may be a mechanism to deactivate the enzyme if the substrate is difficult to oxidize. ~ I ~ to LA
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Excess Water Formation • The 3rd reduction step is apparently very fast - most reactions show some water formation - the reduction rate must be similar to substrate oxidation • The interactions with reductase may be determined by this rate. Cytochrome P450 Mediated Toxicities • Although most P450 oxidations promote elimination (detoxification), occasionally more toxic or mutagenic intermediates are formed. • This may be expected due to the high energy of the active oxygenating P450 intermediate. • Conjugating enzymes are involved in the detoxification of these reactive intermediates. 9 0
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9 N- and O-Dealkylation '~ L'O-+I-r R-OH Ether Hemlacetal or hemiketal Alcohol Carbonyl moiety ' F 4~ IRr-N-C H - ~. Rt-NH + -C , r H¢ L Rs Re TertlaryAmine Carbinolamine SeconoaryAmine Carbonyl moiety • Radical SdHf Reaction Type kcaYmak HzN-CHz CHy-O-CH-CHa CH3-CH-CHa CH3-CHj CHy 17.3 N•deelkylation 19.6 benzylic hydroxylation ?6.6 0-deafkyfadon TI.7 allphatic hydrakylotion 28.6 aliphatic hyoroxyletinn 33.0 orhytlroxylaticn Increasing accumnce of motabollem M CA C-j C6 ti, ~10
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! Predicting the Toxicity of Nitriles CH3-CH2-CH2 CN r p cx NHZ CHZ CHZ CHZ CN kaecrr =ka( ka 1 ka+k@+ 'ky 0
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0 Protein nucleophile x Z+ Protein-Z Lipophilic Electrophilic ~' DNA-Z Xenobiotic Metabolite DNA nucleophile • CO tl'7
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0 Predicting the Toxicity of Nitriles 0.5 • 0.0 0 m G J W ~ 9 ~ -1.0 9 9. -1.5 • . -2.0 ~ -2.0 -1.5 -1.0 -0.5 0.0 0S experimantal lag(1/LD50) Predicting In Vivo Metabolism of Anesthetics -1 5 0 In IFlp.v = 42.8 -1.56(H.ct) R2 .0.86 •eavoeurane •enfi0rans .desflurana 24 25 26 27 28 pradlcted H.n(kcai/mole)
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0 i Nomenclature for the Cytochrome P450s CYP 2 C 9 Cytochrome ~ P450 ~ >40% sequence homology ~ I_. <3% variation >55% sequence homology The CYP1A Subfamily • CYP1A1 - is not found in human liver; found in extrahepatic tissues (induced in the lungs of smokers) • CYP1A2 - is found in human liver - is important in drug metabolism - activates several types of compounds, including PAHs, aromatic amines, and nitrosamines - 0
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0 6' The CYP2E Subfamily ;~ ~ ~ J,w~t`a>> • CYP2E1 - is important for the metabolism of small u~'~' z5 y molecules Olv~ el~ - is inducible by ethanol, acetone, isoniazid, ~ Q~>% , acetone and other compounds ~l ~5}~f - is involved in carcinogenesis t • nitrosamines, acrylonitrile, vinyl halides, benzene, etc. l M11 wU) The CYP3A Subfamily CYP3A4 ( - is very imnortant in human drug metabolism i - is the major form in the liver, ktdney, and GI {" ~IT~y Itlact -' ~'~~ - metabolizes a wide variety of hydrophobic 4 substances • substrates can be very large, e.g. erythromycin (MW=734) and cyclosporine (MW 1201) - is inducible by glucocorticiods and phenobarbital - metabolically activates Aflatoxin Bt
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! (P°1450)3 .+ QZ + NADPH + H~ = (PV 450)':ZS NADP' + H20 (PY450)':O NADP' + Hz0 = (P"450)3 + ROH RH O2 + NADPH + H+= ROH + NADP' + H20 - H Q RCH-X ^ R H-X-~ RCH + HX 33 34 35 R3N -~ RaNY0 36 37 RzS -~ RzSvp- RsSAO 36 39 40 ~ _ D D O : u \ / ( \ METABOLIC BIOACTIVATION OF B@2O(e)PYRENE \ 50 DNA \ METABOLIC ACTIVATION OF AFLATOXIN D oCN3 53 No` Y y ~ ~oH P46 52 c
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0 The Vmax of a P450 oxidation is determined by- - The rates of reduction • rate limiting for the catalytic cycle - the amount of hydrogen peroxide release - the amount of excess water formation 9 Cytochrome P450 Catalytic Cycle ROH > (AOH) Fe+3 (RH) Fe*3 O-0H" ~t, ' F (qH) Fe+3 Hp0 G L (RH)Fe+30•0" (RH) Fe+2 0
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Metabolic Formation of Chemically Reactive Species I. Overview A. Historical perspectives B. Phase I transformations - functionalization C. Phase II transformations - conjugation D. Consequences in terms of biodisposition of xenobiotics II. Bioactivation vs. Detoxification A. The generation of electrophilic metabolic intermediates B. Nucleophilic functionalities present in biopolymer C. The role of glutathione in bioactivation and detoxification processes Ill. Principal Enzyme Systems A. Microsomal flavin containing monooxygenase (FMO) B. The cytochrome P-450 superfamily of monooxygenase C. Monoamine Oxidases IV. Principal Biotransformation Pathways A. Arene substrates B. Olefinic substrates C. Heteroatom containing substrates V. Selected Examples of Biotransformations Leading to Reactive Electrophilic Metabolic Intermediates (As time permits) A. Polycyclic aromatic hydrocarbons B. Aflatoxins C. Olefins D. Aromatic amines E. Heterocyclic amines/amides F. MAO catalyzed amine bioactivations 1<K 0
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• The CYP2C Subfamily • Several forms are present in human liver • CYP2C9 is the most abundant form - metabolizes a variety of compounds, including coumarins, NSAIDS, phenytoin, etc. - active site may be fairly well defined • CYP2C8 has fewer substrates (taxol) • CYP2C19 metabolizes S-mephenytoin - human polymorphism exits • The CYP2D Subfamily ~ CYP2D6 - is present in human liver - defect results in "debrisoquine polymorphism" - metabolizes hydrophobic aniines - amines are probably protonated or quaternary 0 ~~ - SAR studies suggest that a site 5-7A from the cationic center is hydroxylated 0
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carcinogenesis: implications for human variability in expression and enzyme activity. [Review] [128 refs]. Pharmacogenetics 5: 259-274, 1995. 14. Poulos TL, Cytochrome P450. [Review] [48 refs]. Curr Opin Struct Biol 5: 767-774, 1995. 15. Negishi M, Uno T, Darden TA, Sueyoshi T and Pedersen LG, Structural flexibility and functional versatility of mammalian P450 enzymes. [Review] [34 refs]. FASEB J 10: 683-689, 1996. 16. Nelson DR, Koymans L, Kamataki T, Stegeman JJ, Feyereisen R, Waxman DJ, Waterman MR, Gotoh 0, Coon MJ, Estabrook RW, Gunsalus IC and Nebert DW, P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature. [Review] [270 refs]. Pharnaacogenetics 6: 1-42, 1996. 17. Jones JP, Shou M and Korzekwa KR, Predicting the regioselectivity and stereoselectivity of cytochrome P450-mediated reactions: structural models for bioactivation reactions Predicting the regioselectivity and stereoselectivity of cytochrome P450-mediated reactions: structural models for bioactivation reactions. [Review] [11 refs]. Adv Exp Med Biol 387: 355-360, 1996. 18. Guengerich FP, Ueng YF, Kim BR, Langouet S, Coles B, Iyer RS, Thier R, Harris TM, Shimada T, Yamazaici H, Ketterer B and Guillouzo A, Activation of toxic chemicals by cytochrome P450 enzymes: regio-and stereoselective oxidation of aflatoxin B I. [Review] [38 refs]. Adv Exp Med Biol 387: 7-15, 1996. 19. McKinnon RA and McManus ME, Localization of cytochromes P450 in human tissues: implications for chemical toxicity. [Review] [78 refs]. Pathology 28: 148-155, 1996. 20. Wolf CR, Mahmood A, Henderson CJ, McLeod R, Manson MM, Neal GE and Hayes JD, Modulation of the cytochrome P450 system as a mechanism of chemoprotection. [Review] [24 refs]. IARC Sci Publ 165-173, 1996. 21. Badawi AF, Stern SJ, Lang NP and Kadlubar FF, Cytochrome P-450 and acetyltransferase expression as biomarkers of carcinogen-DNA adduct levels and human cancer susceptibility. [Review] [123 refs]. Prog Clin Biol Res 395: 109-140, 1996. 22. Ingelman-Sundberg M, The Gerhard Zbinden Memorial Lecture. Genetic polymorphism of drug metabolizing enzymes. Implications for toxicity of drugs and other xenobiotics. [Review] [26 refs]. Arch Toxicol Supp119: 3-13, 1997. 23. Puga A, Nebert DW, McKinnon RA and Menon AG, Genetic polymorphisms in human drug-metabolizing enzymes: potential uses of reverse genetics to identify genes of toxicological relevance. [Review] [243 refs]. CritRev Toxicol 27: 199-222, 1997. r .
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DHEA mnuhulHS ~ DHEA Andrmknediwe f- A mdahullis Tswtemne meluhnlLLn l ~ nu[enedinrc ~ -w Eslmne ?-Pmhwvy pvrlWlyu[nneAVnebiretl. i- Pu Ox.Y Dort1eY) ehsnnetlred In nur IvM~olwy. SeepreilrWnvrynauls. ?.CPPSC6 M .
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i Nongenotoxic Carcinogenesis • Possible Roles of the Cytochrome P450s - Steroid biosynthesis • e.g. steroid dependent cancers - Steroid metabolism • altered hormone levels • active hormone metabolites - Xenobiotic metabolism • hormone disrupters ! Are the Drug Metabolizing Enzymes Important in Steroid Metabolism? • The same isozymes that metabolize foreign compounds metabolize steroids ~, - Possible functions: • steroid clearance • active metabolites • none r!~' 1~~ ~ ,1~ J V
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0 Variation in Cytochrome P450 Activities • Induction ~ - CYP1A1/CYPIA2 -induced in smokers ~~ Yx - CYP2E1 -induced by ethanol, isoniazid, etc. - CYP3A4 -induced by corticosteroids, rifampin, phenobarbital • Genetic polymorphisms - CYP2D6 - poor metabolizers of debrisoquine - CYPIA2 -variations in caffeine metabolism - CYP3A5 -present in 15% of the population - CYP2C19 -S-mephenytoin hydrolylase - others??
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0 Genetic Polymorphisms • CYP1A2 - Three phenotypes- slow, medium, and fast metabolizers - Genetic sequencing of the population shows no differences in coding or regulatory sequences • suggests transcriptional or translational regulation - Increased bladder cancer susceptibility for slow NAT-2, rapid 1A2 genotype - Increased colorectal cancer susceptibility for rapid NAT-2, rapid lA2 genotype Genetic Polymorphisms • CYP2D6 - Several mutant alleles are involved - marked ethnic differences exist - 1-10% poor metabolizers - Possible link between rapid metabolizers and lung cancer - Poor metabolizers may develop a less aggressive form of bladder cancer t~ W ~ ~
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GSH-ETHYLENE DIBROMIDE ADDUCT NUCLEOPHILIC DNA RFOF~ 0II \CXaC"~CiX - 9rCHaCNaBr H. `t \CNaSH NHCH~COCH NHS H. I q, /C OII ~ C \C"aCXaCNH Nb N.I /CXa Z CHaS` ~ \IXa NHCHrCOOH Amino Acids Bearing Nucleophilic Functionalities HHI HHI N~~\CHrSlI `~' \CHrCHrCHiCH~JHr P1~' 'fl 1 2 ~ H HN/ /FI NH ' \CHrCHZCHSNHCNHr Ifl 3 HOOC HOOC H, ~ ~ o / \LHaL"iC I H HOOI p X. C ~2'~ \C1b5C"aCHaB~ NHCH~COON S{mplNl.d poprotoln portion H.rr» (protoporphyrln IX) portion with "Activ.t.d Onypqri" SubMtrtN badlng sIto NHp H•,_ I HOOCJkCHZCHZCONH E* HOOCCH2HNOC"ti CHZSH 16 (GSH) 0 NHCOCH3 GS•E---- H.... I 17 ~0208228 HOOC,~CHZS-E 18 112 NADPH Fe3*--- Fe3+(RH) ROH ---,A (RH)Fe3+(O) N z ° 112 NADP` (RH)Fe2+ (RH)Fe2+(O2)
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R'CO R"CO ~OCH3 lfOCH3 H__~Y 'I OR /Rll-\H,~~SOP; or ~a sa8~zs 7 1 \ N BIOACTIVATION OF ACETYLAMINOFLUORENE OCH3 ~OCH3 N+ + ~ ~ AONUCT FD FN~TIO~MINOFNUCRENETNATION OCH3 I /V ~JHi'-( ~ s DNK TI.+~ NHZ coCw ~ N^-T NITROGEN MUSTARDS ALKYLATION PATHWAY FOR NITROGEN MUSTARDS RESONANCE STABILIZATION OF CYCLOPHOSPHAMIDE 0
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0 The Cytochrome P450 Enzymes in Chemical Carcinogenesis I. Cytochrome P450 Structure and Function A. General Function B. Catalytic Cycle C. Active Site Interactions II. Cytochrome P450 Subfamilies involved in Drug Metabolism A. The CYPIA Subfamily B. The CYP2D Subfamily C. The CYP2E Subfamily D. The CYP3A Subfamily III. Variations in Cytochrome P450 Activity A. Induction and Inhibition B. Genetic Polymorphisms IV. Non-genotoxic Carcinogenesis A. Steroidogenic P450s B. Estrogenic Compounds and Breast Cancer 0
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Metabolic Formation of Chemically Reactive Species Interest in the metabolic fate of xenobiotics stems in part from the role that they play in the mediation of a variety of toxic effects Including call transformation and cell death. This presentation is an attempt to summarize the key metabolic transformations that are responsible for the formation of chemically reactive intermediates that mediate these toxic effects. This presentation will follow the attached outiine. Mammalian enzyme catalyzed reactions of lipophilic xenobiotics are divided into "Phase I transformations" that introduce a functional group followed by "Phase II transformations" in which the initially formed metabolite Is converted into a polar conjugate such as a glucuronide or sulfate derivative. The resulting polar metabolite is readily excreted by the kidney. Although the above metabolic sequence generally serves to protect mammals against the toxic effects of xenobiotics, the structural characteristics of specific agents may lead to the formation of electrophilic intermediates which are capable of forming covalent adducts with nucleophilic tunctionatities present on biomacromolecules including proteins, RNA and DNA. Mechanisms such as those involving the reaction with glutathione, generally protect against the toxic effects of electrophllic metabolic intermediates, but even glutathion conjugation can lead to toxic outcomes. The principal enzymes involved in the Phase I biofunctionalization reactions Include the microsomal flavin containing monooxygenases and for selected amines, the monoamine oxidases. The most extensively studied and Important group of enzymes in the metabolic bioactivation of xenobiotics is the cyctochrome P-450 super family of hemoproteins. Detailed mechanistic information is available on such key reactions as the cyctochrome P-450 catalyzed oxidations of arenes, olefins and heterocyclic systems. This presentation will attempt to provide a range of examples of how the cyctochrome P-450's convert a variety of lipophilic xenobiotics to electrophific Intermediates and the nature of the interactions of these intermediates with biomacromotecuies. The examples will include such compounds as polycyclic aromatic hydrocarbons, the aflatoxins, arylamines, pyrrolizidine alkaloids, various olefins and other amine and amide containing systems. Bioactivation pathways involving Phase II transformations also will be discussed. kkC
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0 THE CYTOCHROME P450 ENZYMES AND CHEMICAL. CARCINOGENESIS KENNETH KORZEKWA w
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Are the Drug Metabolizing Enzymes Important in Steroid Metabolism? • Possible functions: • steroid clearance - cimetidine-estrogeninteraction • active metabolites - 16a-OH-E1, catechol estrogens • Other unknown functions - CYPIA2 - CYP2C9 R144C mutation • Steroid Biosynthesis 17-OH-Pregnenolone z- DFoc~mn ~ir,t, 11-Deoxyeortisol . r ( iii,i,l~C~n~~.~ Corfisol 18OH C~.1ra.~~.<<r~~ae Y Aldosterone tA 1 69, 101~- ii 7 iA~ ~d~.Pi r,un ~d-j;- HEA Audrosfenedione '. ~ Estrone . Testosterone " Estradiol 1 ~ t~h ~.J Cd9 J 17-OH-Progesteroi)e
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• 0 References 1. Gonzalez FJ, Crespi CL and Gelboin HV, DNA-expressed human cytochrome P450s: a new age of molecular toxicology and human risk assessment. Mutat Res 247: 113-127, 1991. 2. Gonzalez FJ and Nebert DW, Evolution of the P450 gene superfamily: animal-plant 'warfare', molecular drive and human genetic differences in drug oxidation. Trends Genet 6: 182-186, 1990. 3. Gonzalez FJ, Molecular genetics of the P-450 superfamily. Pharmacol Ther 45: 1-38, 1990. 4. Robinson RC, Nagata K, Gelboin HV, Rifkind J, Gonzalez FJ and Friedman FK, Developmental regulation of hepatic testosterone hydroxylases: simultaneous activation and repression of constitutively expressed cytochromes P450 in senescent rats. Arch Biochem Biophys 277: 42-46, 1990. 5. Idle JR, Armstrong M, Boddy AV, Boustead C, Cholerton S, Cooper J, Daly AK, Ellis J, Gregory W and Hadidi H, The pharmacogenetics of chemical carcinogenesis. Pharinacogen 2: 246-258, 1992. 6. Harris CC, Chemical and physical carcinogenesis: advances and perspectives for the 1990s. Cancer Res 51: 5023s-5044s, 1991. 7. Tennant RW, Elwell MR, Spalding JW and Griesemer RA, Evidence that toxic injury is not always associated with induction of chemical carcinogenesis [see comments]. Mol Carcinog 4: 420-440, 1991. 8. Osborne MP, Bradlow HL, Wong GYC and Telang NT, Upregulation of estradiol C16a-hydroxylation in human breast tissue: A potential biomarker of breast cancer risk. JNCI85: 1917-t920,1993. 9. Kadlubar FF, Biochemical individuality and its implications for drug and carcinogen metabolism: Recent insights from acetyltransferase and cytochrome P4501A2 phenotyping and genotyping in humans. Drug Metab Rev 26: 37-46, 1994. 10. Ortiz de Montellano PR, The 1994 Bernard B. Brodie Award Lecture. Structure, mechanism, and inhibition of cytochrome P450. [Review] [61 refs]. Drug Metab Dispos 23: 1181-1187, 1995. 11. Park BK, Pirmohamed M and Kitteringham NR, The role of cytochrome P450 enzymes in hepatic and extrahepatic human drug toxicity. [Review] [369 refs]. Pharmacol Ther 68: 385-424, 1995. 12. Femandez-Salguero P and Gonzalez FJ, The CYP2A gene subfamily: species differences, regulation, catalytic activities and role in chemical carcinogenesis. [Review] [56 refs]. Pharmacogenetics 5: Spec No:S123-8, 1995. * 13. Eaton DL, Gallagher EP, Bammler TK and Kunze KL, Role of cytochrome P4501A2 in chemical
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! CURRICULUM VITAE Name: Kenneth R. Korzekwa July 24, 1995 Date and Place of Birth: December 25, 1957; Hobbs, New Mexico Citizenship: United States Education: Dec. 1980 - B.S. (Chemical Engineering) New Mexico State University March 1987 - Ph.D. (Medicinal Chemistry) University of Washington (Thesis: Theoretical and Isotope Effect Studies on Cytochrome P-450 Mediated Aromatic Oxidation.) ! Brief Chronology of Employment: 1980-1981 - Chemical Engineer, Union Carbide Corp., Charleston, WV. 1981-1987- Graduate Research Assistant: Dept. of Medicinal Chemistry, University of Washington, Seattle, WA. 1987 - Staff Fellow, NIH, NHLBI, Laboratory of Chemical Pharmacology 1987-1989 - NIH, Pharmacology Research Associate Training Program (PRAT Fellow) 1989-1990- Senior Staff Fellow, NIH, NHLBI, Laboratory of Chemical Pharmacology 1990-1994- Senior Staff Fellow, NIH, NCI, DCE, Laboratory of Molecular Carcinogenesis 1995-Date- Visiting Associate Professor, Center for Clinical Pharmacology; Director, Drug Discovery Program, University of Pittsburgh Editorshins: Editorial Review Board, Journal of Biochemical Toxicology. Research Interests: Mechanistic studies in drug and steroid metabolism; The use of theoretical methods and stable isotopes to probe chemical and biological systems. Present Home Address: 6526 Dalzell Place Pittsburgh, PA 15217 co 412-421-9344 w ~ (-~j rn 0 w ~ fV
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0 Cytochrome P450s and Chemical Carcinogenesis- The cytochrome P450s are involved in: Genotoxic carcinogenesis- activation of procarcinogens to carcinogens. Nongenotoxic carcinogens- steroid biosynthesis steroid metabolism xenobiotic metabolism 0 Cytochrome P450s Over 300 sequenced to date 40 families in all organisms 12 families in mammals Xenobiotic metabolizing P450s: CYP1 (2 subfamilies) CYP2 (7 subfamilies) CYP3 (2 subfamilies) CYP4 (3 subfamilies) A high degree of species differences in number, catalytic activity and regulation. © © THE CYTOCHROME P450S ARE A SUPERFAMILY OF ENZYMES THAT METABOLIZE A WIDE VARIETY OF ENDOGENOUS AND EXOGENOUS COMPOUNDS. The Functions of Cytochrome P-450: 1. Steroid Biosynthesis/Degradation. 2. Prostaglandin and Leukotriene Biosyn./Degrad. 3. Drug Metabolism. 4. Xenobiotic Bioactivation. 5 111208m SOME CYTOCHROME P450S APPEAR TO HAVE EVOLVED SPECIFICALLY FOR THE METABOLISM OF XENOBIOTICS THIS TASK IS ACCOMPLISHED BY: A superfamily of enzymes Broad and overlapping substrate specificity A very reactive oxygenating species
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• FORMATION OF THE GLYCINE CONJUGATE OF BENZOIC ACID COOH µ~NCqCOOH ~ CONHCHzC00 RH Phase i~- ROH Phase i~- HO OR 0 OENERAL SUMMARY OF PHASE I AND PHASE 11 METAEOLIC PATH WAYS PHASE I OR FUNCTIONALIZATION REACTIONS O.Waw Narnnn. Ouaalon Ol.romac mu~nia. oae.eon ul a.nn. o.un~an .1 o.nryl/c..l0<u~mn .mm. w urwn amm. upna lo urconW uq knln.. O.qaliGn p Ylplrac u~G a~lGyqlc C4~rl.bnn Qlb.llpi IMDMnV V~1~"Mlxn.lom.ya.m Ca1ppIFn11r4yM.y.l.ma(.Iipn.11C a/M.romWc 4mn..~ mduE.. NU..IYYIaW11.0aM.11.. CMmnvtlon. Na.qanma~nn. NAyCrCryYYn.n). LvnGAaayp.n Mlmn(Oa.YkyLEOn) L'ypqHYlNr aya1MM ($bakyy(Ipl. $-Y)Y1tlR anG E~ulluratlpl) O.n.nor/ a alcdWa uM a1G.I~C~ pM1~ ml `~,a~'WYqlla oa.tapv. MCIb1N a.ak.ellw N.a1mu FbuCTnn ul alOaryQe a/1E kNOrro. R.uuninn al mko uq aro mmVaunG. MiYYparN]ui raGUt1M r.KAdi Rprul,tlc pa.Ctlar~ MWray.n ul Ym.n.na .n.u.. NyNan4n a 1GOtIUS afq YRN oatla Ey ~pJ//.la nrora PHASE II OR CONJOGATION REAGTIONS GWwrdxc.cq o.yup.nm Suha\a Co~HrqallRr r IUPa1MnN11~PhO/M.OkIWUM.MUENraIHn4 cm. OWNeIw u m.ruPMb KM cunryiacn Aq./Irar MWryYYM Figure 4. The genenl structurd features of a mammalian ncphron. POTENTIAL OXIDATIVE METABOLIC FATE OF XENOBIOTICS CH3C NHCOCH3 Ph 1-12'~ ~ 1 Phesell LO2Q8228 3 efel Hippuric Acid 2 NHCOCH3 NHCOCH3 RH = ROH RO-X I --- RH(O) I MACROMOLECULAR ADDUCTS
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Ospecific Binding Interactions ~ CYTOCHROME P450 MEDIATED TOXICITIES'"' Although metabolism by the cytochrome P450s usually promotes elimination (detoxification), occasionally more toxic and/or mutagenic intermediates are formed. This may be expected due to the high energy of the active oxygenating P450 intermediate. Some xenobiotic conjugating enzymes are involved in the detoxification of these reactive intermediates, i.e. epoxide hydrolase and glutathione transferase. dD The Vmax of a P450 oxidation is determined by: 1. The rates of reduction 2. The amount of hydrogen peroxide release 3. The amount of excess water formation.
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Benzo[a]pyrene Activation ! HO'" OH [ol P450 [07 P450 HO"' H20 Epoxide Hydrolase Diol-epoxides formed in the metabolism of B[a]P 7,8-dihydrodiol by cDNA expressed CYPs in Hep. G2 cell line. 4500 4000 3500 3000 2500 2000 500 450 400 350 300 250 200 150 100 50 0 W 3 Q U U N N N L L t L E N Q E N / DE-I EM DE-11
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0) The "catalytic" steps for the xenoblotic metabolizing cytochrome ~ ENZYMOLOGY OF THE CYTOCHROME P450S Are the Cytochrome P450s good enzymes? Most enzymes: metabolize one substrate with one mechanism fast and efficiently. ROH P450s are probably associated with oxygen activation. 2e- + 2H+ ~Fe~ + 02 SR SR H20 This active oxygen species is very reactive and can metabolize most functional groups. -hydrogen abstraction, even from very deactivated groups, e.g. alkanes. -addition to unsaturated functionalities, e.g. alkenes and . aromatic compounds. Cytochrome P450 Catalytic Cycle (ROH)Fe+3 2e-+2H+ (R.H)Fe 30 Many cytochrome P450s: metabolize many substrates with multiple mechanisms slowly and inefficiently. OD (RH)Fe+2 0 HO (RH)Fe+3O-OH- Fe+3 H20 e +H+ J (RH)Fe+30-O% 9 Specific Binding Interactions (e.g. Steroidogenic P450s)
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0 HYDROGEN PEROXIDE RELEASE Hydrogen peroxide release may result from too many water molecules in the active site. -only substrates which can exclude water from the active site can be efficiently metabolized. This release may prevent the generation of the active oxygen in the absence of a substrate. e EXCESS WATER FORMATION Excess water formation: - is incompetition with substrate oxidation -is initiated by the introduction of a third electron. This may be a mechanism to deactivate the active oxygen when the substrate is difficult to oxidize, preventing oxidation of the enzyme. 8G20B22g Nomenclature for the Cytochrome P450s 0% CYTOCHROME P450 >40% SEQUENCE HOMOLOGY CYP 2 C 9 L_ >3% VARIATION / ~ >55% HOMOLOGY (MAMMALIAN) THE CYP1A SUBFAMILY CYP1A1: - is not found in human liver is but found in extrahepatic tissues (induced in the lungs of smokers). CYP1A2: - is found in human liver. - is important in drug metabolism. - is thought to be responsible for the metabolic activation of several types of compounds.including polycyclic aromatic hydrocarbons, aromatic amines, and nitrosamines. 9
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~ 0 Wang, M: Y. and Liehr, J.G. (1994). Identification of fatty acid hydroperoxide cofactors in the cytochrome P450-mediated oxidation of estrogens to quinone metabolites. Role and balance of lipid peroxides during estrogen-induced carcinogenesis. J. Biol. Chem. 269, 284-291. Han, X. and Liehr, J.G. (1994). DNA single-strand breaks in kidneys of Syrian hamsters treated with steroidal estrogens: Hormone-induced free radical damage preceding renal malignancy. Carcinogenesis 15, 997-1000. CO W C~~! CJO ~ CD Crl hJ ~
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MECHANISMS OF LIVER AND KIDNEY TOXICITY CURTIS D. KLAASSEN
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SELECTED REEERENCES TO THE CYTOCHROME P450 ENZYMES THROUGH i992"' The references given below refer to the cytochrome P450 enzymes and are listed in chronological order. Citations to review articles, books and book chapters are given in bold type. 01945. Miller JA, Baumann CA. The carcinogenicity of certain azo dyes related to p- dimethylaminoazobenzene. Cancer Res 1945;5:227-234. 1947. Miller EC, Miller JA. The presence and significance of bound aminoazo dyes in the livers of rats fed p-dimethylaminoazobenzene. Cancer Res 1947;7:468-480. 1953. Meechan RJ, McCafferty DE and Jones RS. 3-Methylcholanthrene as an inhibitor of hepatic cancer induced by 3'-methyl-4-dimethylaminoazobenene in the diet of the rat: A determination of the time relatioships, Cancer Res 1953:802-806. 1957. Conney AH, Miller EC, Miller JA. Substrate-induced synthesis and other properties of benzopyrene hydroxylase in rat liver. J Biol Chem 1957;228:753-766. 1958. Miller EC, Miller JA, Brown RR and MacDonald JC. On the protective action of certain polycyclic aromatic hydrocarbons against carcinogenesis by aminoazo dyes and 2acetylaminofluorene, Cancer Res 1958;18:469-477. 1961. Arcos JC, Conney AH, Buu-Hoi NP. Induction of microsomal enzyme synthesis by polycyclic aromatic hydrocarbons of different molecular sizes, J. Biol. Chem. 1961;236:1291-XXX. 1962. Magee PN, Farber E. Toxic liver injury and carcinogenesis. Methylation of rat liver nucleic acids by dimethylnitrosamine in vivo. Biochem J 1962;83:114-124. 1965. Huggins CB and Pataki J. Aromatic azo derivatives preventing mammary cancer and adrenal injury from 7,12-dimethylbenz(a)anthracene, Proc Natl Acad Sci USA 1965;53:791-795. 1965. Omura T, Sato R, Cooper DY, Rosenthal 0, Estabrook RW. Function of cytochrome P-450 of microsomes. Fed Proc 1965;24:1181-1189. 1966. Lotlikar PD, Scribner JD, Miller JA, Miller EC. Reaction of esters of aromatic N-hydroxy amines and amides with methionine in vitro. A model for in vivo binding of amine carcinogens to protein. Life Sci 1966;5:1263-1269. 1966. Miller EC, Miller JA. Mechanisms of chemical carcinogenesis: nature of proximate carcinogens and interactions with macromolecules. Pharmacol Rev 1966;18:805-838. 1968. Wattenberg LW and Leong JL. Inhibition of the carcinogenic action of 7,12dimethylbenz(a)anthracene by beta-naphthoflavone, Proc Soc Exp Biol Med 1968;128: 940-943. 1968. Wheatley DN. Enhancement and inhibition of the induction by 7,12dimethylbenz(a)anthracene of mammary tomours in female sprague-dawley rats, Brit 7 Cancer 1968;22:787-797. 1971. Williams RT. Detoxification mechanisms. 2nd ed. New York: John Wiley and Sons, 1971. 1971. Yamamoto RS, Weisburger JH and Weisburger EK. Controlling factors in urethan carcinogenesis in mice: Effect of enzyme inducers and metabolic inhibitors, Cancer Res 1971:31:483-486. 1972. Lu AYH, Levin W. Partial purification of cytochrome P-450 and P-448 from rat liver microsomes. Biochem Biophys Res Commun 1972;46:1334-1340. 1973. Borgen AO, Darvey H, Castagnoli N, Crocker TT, Rasmussen RE, Wong IY. Metabolic conversion of benzo[a]pyrene by Syrian hamster liver microsomes and binding of metabolites to deoxyribonucleic acid: J Med Chem 1973;16:502-506. 1975. Ames BN, McCann J and Yamasaki E. Methods for detecting carcinogens and mutagens with the sahnonella/mammalian-microsome mutagenicity test, Mutation Research 1975;31:347-364. 1976. Huberman E, Sachs L, Yang SK, Gelboin HV. Identification of mutagenic metabolites of benzo[a]pyrene in mammalian cells. Proc Natl Acad Sci USA 1976;73:607-611. 1977. Jerina DM, Yagi H, Hernandez O. In: Jollow DJ, Kocsis JJ, Synder R, Varinio H, eds. Biological reactive intermediates. New York:Plenum Press;1977:371-378. 1977. Poland A, Kende A. The genetic expression of aryl hydrocarbon hydroxylase activity: evidence for a receptor mutation in nonresponsive mice. In:Hiatt HH, Watson JD, Winsten JA, eds. 06 Origins of human cancer. New York: Cold Spring Harbor Laboratory,1977:847-867. U-3 1978. Kouri RE, Rude TH, Joglekar R, Dansette PM, Jerina DM, Atlas SA, Owens IS, Nebert DW. C"i 2,3,7,8Tetrachlorodibenzo-p-dioxin as cocarcinogen causing 3-methyicholanthrene-initiated subcutaneous w tumors in mice genetically "nonresponsive" atAh locus, Canc. Res. 1978;38:2722-2783. ci 1978. Miller EC. Some current perspectives on chemical carcinogenesis in humans and experimental rrJ ~animals: presidential address. Cancer Res 1978;38:1479-1496. C:? 1979. Cohen GM, Bracken WM, Iyer RP, Berry DL, Selkirk JK and Slaga T7. Anticarcinogenic effects of °~ 2,3,7,8-tetrachlorodibenzo-p-dioxin on benzo(a)pyrene and 7,12dimethylbenz(a)anthracene tumor initiation and its relationship to DNA binding, Cancer Res 1979;39:4027-4033. 1980. White RE, Coon MJ. Oxygen activation by cytochrome P450. Annu Rev Biocheto,1980;
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• The Cytochrome P450 Enzymes and Chemical Carcinogenesis The cytochrome P450s are a superfamily of monooxygenases involved in the metabolism of both exogenous and endogenous compounds. These enzymes play a role in both genotoxic and nongenotoxic chemical carcinogenesis. The xenobiotic metabolizing P450s show broad and overlapping substrate specificities which allows for the metabolism of a wide variety of hydrophobic compounds. The active oxygen generated by this enzyme is very reactive and can react with several different kinds of functional groups. However, this reactivity sometimes results in the formation of unstable reactive intermediates, which can react with macromolecules and result in toxicity and mutagenicity. Several human cytochrome P450s appear to be important in the activation of procarcinogens. In the CYP1A subfamily, 1A1 and 1A2 are induced in smokers and can activate several classes of compounds, including polycyclic aromatic hydrocarbons, aromatic amines. CYP2E1 preferentially metabolize and can activate small compounds and CYP2D6 metabolizes cationic amines. CYP3A4 metabolizes a wide variety of molecules including very large molecules such as cyclosporin. Enzyme levels and activities can vary greatly between different individuals. These differences can be due to different states of induction and genetic polymorphisms. Induction of human P450s include increased CYP1A in smokers, CYP2E1 after treatment with ethanol or isoniazid, and CYP3A4 with corticosteroids or phenobarbital. Genetic polymorphisms include defects in CYP2D6 and variations in CYP1A2 activities. Poor 2D6 metabolizers may have a decreased incidence of lung cancer and rapid 1A2 metabolizers may have increased ~ incidences of bladder and colorectal cancers. The cytochrome P450s may be involved in nongenotoxic carcinogenesis. Several P450s are involved in steroid biosynthesis and metabolism. Cumulative estrogen levels are postulated to be an important determinant of breast cancer. P450 inhibitors have been reported to alter estrogen levels in man. Some exogenous compounds and their metabolites have estrogenic activity. It has been suggested that a general increase in estrogenic activity may be responsible for the increases in breast and testicular cancers. Finally, 16a- hydroxyestrone formation, a P450 metabolite of estrogen, may be a determinant for breast cancer. 00 CN W ~ CO O W .. .~.
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0 THE CYP2D SUBFAMILY CYP2D6: -is present in the liver and very important in drug metabolism. -defect results in "debrisoquine polymorphism" -primarily metabolizes hydrophobic amines. -amines are probably protonated or quaternary. -SAR studies suggest that positions 5-7 k from the cationic center are hydroxylated. THE CYP2E SUBFAMILY CYP2E1: -is important in the metabolism of small molecules. 0 -is inducible by ethanol , isoniazid, acetone, and other compounds. -is involved in certain carcinogenis activities, i.e. nitrosoamines, acrylonitrile, vinyl halides, and benzene. • THE CYP3A SUBFAMILY ~ CYP3A4: -is very important in drug metabolism. -is expressed in liver, kidney and GI tract. -metabolizes a wide variety of hydrophobic substrates. -substrates can be very large i.e. erythromycin (MW 734) and cyclosporin (MW 1200). -is inducible by glucacorticoids and phenobarbitol. -metabolically activates aflatoxin Bl, nitroaromaties, etc. CYP3A5: -expressed in the liver in only 15% of the population. -found in the placenta. -substrate specificity similar to CYP3A4. CYP BA7: -major fetal liver P450 -not found in adults except in placenta. -metabolizes dehydroepiandrosterone sulfate. CYP3A4 Activation Activation- The metabolism of some substrates is increased in the presence of certain compounds. Autoactivation- Nonlinear Michaelis-Menten liinetic: Appears to be associated with two molecules simultaneously occupying the same active site. Mutagenic activity of procarcinogens can also be increased by 3A4 activators. 6L209228
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Varia*s in Cytochrome P450 Activities (~Jg - _ Genetic Polymorphisms Induction: CYP1A1/ CYP1A2 - induced in smalcers. CYP2E1 - induced by ethanol, isoniazid, etc. CYP3A4 - induced by corticosteroids, phenobarbital. Genetic Polymorphisms: CYP2DG - poor metabolizers of debrisoquine. CYP1A2 - variations in caffeine metabolism. CYP3A - present in 16% of the population. CYP2CI9 - S-mephenytoin hydroxylase (?). Others- ??? CYP2D6 Several mutant alleles are involved. Marked ethnic differences exist. Usually 1-10% poor metabolizers. Possible link between rapid metabolizers and lung cancer. Poor metabolizers may develop less aggressive forms of bladder cancer. Carcinogenesis and P450 Induction and Inhibition a Xenobiotics are often metabolized by more than one P450 to several products. Competition between activating and detoxifying pathways may be important. P450 inducers and inhibitors can affect the relative contributions of the different metabolic pathways. -Can P450 inducers or inhibitors be used ta prevent toxicities? Genetic Polymorphisms CYP1A2 Three phenotypes- slow, medium, and rapid metabolizers. Genetic sequencing of the populations show no difference in coding or regulatory sequences, suggesting differences in transcriptional or translational regulation. Increased blader cancer susceptibility for slow NAT-2, rapid 1A2 populations. Increased colorectal cancer susceptibility for rapid NAT-2, rapid IA2 populations.
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0 Ol NEPATOTOXICRY Hepatotoxiciry or liver Injury Is one of the most common toxic effects produced by chemicals. Hepatotoxic chemicals are dassified as those which induce liver injury predictably and non-predictabiy. Examples of chemicals and the reasons for placing them in these categories will be discussed. Molecular mechanisms of hepatotoxioity are lipid peroxidation, covalent binding, trapping of endogenous substrates and redox cyciing. CO w w 00 ,~ o ~ w w KB ~
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• IV. CLASSIFICATION OF HEPATOTOXIC COMPOUNDS (in abbreviation) A. T_ype I: Compounds which induce liver injury predictably 1. Produce necrosis and/or fatty changes 2. Produce cholestasis B. Typ.g,ll: Compounds which induce liver injury nonpredictably r 1. Produce parenchymal injury 2. Produce cholestasis C. Compounds which produce hepatic cancer V. CLASSIFICATION OF HEPATOTOXIC COMPOUNDS (in detail) A. Properties of compounds which induce type I liver injury (predictable) 1. Exhibit a distinctive histopathologic pattern for any given hepatotoxin 2. Vary in severity in relation to the dose 3. Can be elicited in most if not all individuals 4: Are reproducible in experimental animals CHor 00"' 5. Appear after a predictable and usually brief latent period following exposure ( Z dv,~) B. Subclassification of type I hepatotoxins 1. Compounds acting to produce necrosis and fatty 00 metamorphosis of the liver parenchyma w . K3
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! ! NEPHROTOXICITY The kidney plays an important role in the elimination of chemicals from the body. As a result, many chemicals ooncentrate In the kidney and are biotransformed there to produce toxicity. The mechanisms of nephrotoxicity produced by metals, halogenated hydrocarbons, hcxachlorobutadieno, analgesics, enesthetics, antibiotics, and mycotoxins will be tliscussed. w w w CO ~ o w w 0 KC
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SOME CHARACTERISTICS OF CYP1A2 (1) Calelyzes the bloaetivatlon of arylamines, arylamldes, azaheterocyciie food muta~gens end promulegens present In cigarette smoke condensae. (2) mCata ark:r for CYP7 ~ ~egviry tion of caffeine which is a good in vlvo (3) 1s expressed in human liver but levels vary signlacanny (»10 fold) (4) LIver enzyme Is Inducd by polycyclic aromatic hydrocarbons, olgarette amoks, eNdferous vegetables and charbrolld mast. (5) Is Inhlbitd by a-nephthoeevone and fumfylline. POSSIBLE CONSEQUENCES OF CYP1 A(NDUCTION (1) Increesed hepstotoxldty following exposure to compounds such acetsminopMn. (2) bloacp~vadt dbyCYPlA1arldlorCYP1A2Grcinogenswhichere (3) Protection against toxic chemicals (such as the sflatoxins) which ara detoxl/Nd by CYP7 Af and/or CYPt A2. INDU -TION OF CYP1A SUBFAMILY BY OMEPRAZOLE (1) Studies with primary cultures of human hepatocytes. (2) Studies with liver microsomes obtained patients. (3) Relationship to Alrrespons(veness. (4) In vivo measurements. 0 yOGOS22S MUTAGENICITY VS TUMORIGENICITY Many tumorlgenic chemicals are mutagenlc In short term genotoxicity essays only In the presence of mlcrosomal activating enzymes. In some Instances the genotoxic potential of a chemical may be missed because the bacterlel assay Is lacking Phese 11 enzymes required to generate the "ultimate" bloalkylatmg agent. In some Instances a chemical msy be genotoxlc but have little tumorlgenlc polentlal beeause normal deloxlGCation enzymes (often those that atdyze Phnse N transformations) ere mlasing. Some tumorigens yield negative results In short-term genotoxiciry tests because they cause tumors byep igen®tic mechanisms such as those Invotving Immune suppresslon, hormonal alteration, metabolic imbalana, chronic inflammallon, mltogenesls, proliferation of subcellular orgenells, and decreased fidelity of DNA replication or repalr. INDUCTION OF CYPIA SUBFAMILY AND CHEMICAL TUMORIGENICITY Inductlon of CYPI A11CYP7 A2 In rodents with polycycllc aromatic hydrocarbons, TCDD, p-naphthoflavone, and other agents provides protection egslnst the cerclnogenlc eHects of a variety of chemicals which are known to be bioacllvatd by CYP1A1 andlor CYP7 A2. For example, induction of CYP7 A2 by p-naphlho0avone protects rats from the hepaloc.rcinogenic effecb of aflatoxln. The protective agents must be administered prior to the chemical, that Is In sufficient time to allow enzyme Induction to occur. Treatment with CYP1A Inducers provides protectlon agalnst chemical carcinogenesls in AFFrssponsive mice but not non-responsive mice. Treatment of rats with the SKF 525A, a cytochrome P450 Inhibitor,l hour before carcinogen doslng~ potentlald the tumorlgenkaty of 7,72~Imethyl- benz(a~ nthracane. On ihe other hand,1atnynylpyrena, a mechanism based Inactivators of cytochrome P450 en es, has been shown to block the development of skin tumors by benzo(®~pyrene in SENCAn mice. 0
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Breast Cancer and Estrogenic Compounds Some cancers are hormone dependent (4A% of breast cancers are estrogen dependent). Aromatase is a cytochrome P450 that converts androgens to estrogens. Aromatase inhibitors are being tested for use in breast cancer and benign prostate hyperplasia treatment. 16a-hydroxyestrone, a P450 mediated estrogen metabolite, reportedly binds irreversibly to, and activates the estrogen receptor. Breast Cancer and Estrogenic Compounds Some environmental contaminants or their metabolites have estrogenic activity. This activity is complex and tissue dependent. All compounds cannot be classified as simple agonists or antagonists. Levels of DDE, a DDT metabolite, have have been shown to correlate with breast cancer risk. In addition, catechol estrogens have been implicated in estradiol induced tumorigenesis in hamster liidney. Fig. 1. Constitutive levels of E: C16p-hydroxylation in explant cultures of human mammary tissue. Mammary fat from four low-risk and four high-risk patients (A) or TDLUs trom the same eight patients (0) were used. Radiometric determination of E, CI6a-hydroxylation on six independent explant cultures per patieni is presenred. Mean percent 3 SD-of E3 C36a-hydruxylation per milligram of tissue is corrected for nonspecific tritium exchange from 48-honr incubatinn of /CIRa-`Hj E, without rissue. MF = mammary fat, HR = high-risk, LR = low-risk, and ns = nonsignificant. 2 ~2 0 8 2 2 8 o,.-E"er.- J-,l- 6-2 .PN 0- 1 , r s53
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0 References • I* Harris, C.C. (1991). Chemical and physical carcinogenesis: advances and perspectives for the 1990s. Cancer Res. 51, 5023s-5044s. Tennant, R.W., Elwell, M.R., Spalding, J.W., and Griesemer, R.A. (1991). Evidence that toxic injury is not always associated with induction of chemicai carcinogenesis [see comments]. Mol. Carcinog. 4, 420-440. White, R.E. and Coon, M.J. (1980). Oxygen activation by cytochrome P-450. Annu. Rev. Biochem. 49, 315-356. Coon, M.J., Ding, X.X., Pernecky, S.J., and Vaz, A.D. (1992). Cytochrome P450: progress and predictions. FASEB J. 6, 669-673. Ortiz de Montellano, P.R. (1986). Cytochrome P-450 (New York: Plenum Press). Nelson, D.R., Kamataki, T., Waxman, D.J., Guengerich, F.P., Estabrook, R.W., Feyereisen, R., Gonzalez, F.J., Coon, M.J., Gunsalus, LC., Gotoh, 0., Okuda, K., and Nebert, D.W. (1993). The P450 superfamily: Update on new sequences, gene mapping, accession numbers, early trivial names of enzymes, and nomenclature. DNA Cell Biol. 12, 1-51. Gonzalez, F.J., Crespi, C.L., and Gelboin, H.V. (1991). DNA-expressed human cytochrome P450s: a new age of molecular toxicology and human risk assessment. Mutat. Res. 247, 113-127. Gonzalez, F.J. and Gelboin, H.V. (1994). Role of human cytochromes P450 in the metabolic activation of chemical carcinogens and toxins. Drug Metab. Rev. 26, 165-183. Gorsky, L.D., Koop, D.R., and Coon, M.J. (1982). On the Stoichiometry of the Oxidase and Monooxygenase Reactions Catalyzed by Liver Microsomal Cytochrome P-450. J. Biol. Chem. 259, 6812-6817. Nordblom, G.D. and Coon, M.J. (1982). Hydrogen Peroxide Formation and Stoichiometry of Hydroxylation Reactions Catalyzed by Highly Purified Liver Microsomal Cytochrome P-450. Arch. Biochem. Biophys. 180, 343-347. Grogan, J., Shou, M., Zhou, D., Chen, S., and Korzekwa, K.R. (1993). Use of aromatase (CYPI9) metabolite ratios to characterize electron transfer from NADPH-cytochrome P450 reductase. Biochemistry 32, 12007-12012. Johnson, E.F., Schwab, G.E., and Vickery, L.E. (1988). Positive effectors of the binding of an active site-directed amino steroid to rabbit cytochrome P450 3c. J. Biol. Chem 263, 17672-17677. Schwab, G.E., Raucy, J.L., and Johnson, E.F. (1988). Modulation of rabbit and human hepatic
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0 D. 1. Serum glutamic pyruvic transaminase (SGPT) or alanine amino transferase (ALT) J Serum enzymes Serum glutamic oxalacetic transferase (SGOT) or aspartic amino transferase (AST) 3. Alkaline phosphatase (AP) VI11. CHOLESTASIS A. Classification of cholestasis (chole, gall: stasis, standing still) 1. Extrahepatic a. Cholesterol kept in solution by cholesterol-bile acid micelles b. Gallstones are due to precipitated cholesterol in bile c. Chemicals cause gallstones ! (1) Estrogens t,rwovie-. (2) Clofibrate fi 3,~ 2. , Intrahepatic J B. Electron microscopic changes in intrahepatic cholestasis 1: Canaliculi lose their microville and dilate - normally 1 u in diameter 2. Plugs in the canaliculi 3. Cytoplasm near canaliculi becomes more dense 4. Golgi zone dilates w LIM 5. Increase in S.E.R. w (to C3 K9
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! Nongenotoxic carcinogenesis: Possible roles by the cytochrome P450s: Steroid biosynthesis -e.g. hormone dependent cancers Steroid metabolism -altered hormone levels -active hormone metabolites Xenobiotic metabolism -active xenobiotic metabolites / cholestarol :_ i cp- P~nnnnnnUw~q MI --- - t7-ot+Pregnennlone-. - DHEn n,asc0 a,:~; iLnriicosterone \1 SYDPRODUCT_ MfNERALOCORTiCOID A11dfDShlflBdl ' . EShU119 . ~ ~ _ . .. ~. ~.,Iisd Tes4oatamm -:::- Estraaid , .~. -~-~ cwssow , p. 1^/ 9z rdaM-Hlco~l GLUCGCAR7tCO(D /WDROGEN ESTROGEN 209 0 0
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IX. AN EXAMPLE OF HEPATOTOXICITY PRODUCED BY LIPID PEROXIDATION: CCI, HEPATOTOXICITY ~- A. Biotransformed to trichloromethyl free radical (•CCI,) ~I 1. By P-450 system ~ s k bit l h 2 DDT d h b h t t i it ~, . an p eno ar a en ance epa o ox c y B. Trichloromethyl free radical is chemically very reactive 1. React with hydrogen to form chloroform 2. Can react with itself to form hexachioroethane 3. Can bind nucleic acids (cancer?) 4. Can bind proteins 5. Can peroxidize the polyenoic lipids of the ER and the subsequent generation of secondary free radicals derived from lipids of the membrane 1~ a. Lipid peroxidation destroys ER, results in (1) Decrease in P-450 (2) Decrease in protein synthesis C. Fatty liver: due to a decrease in coupling of the lipid acceptor protein with the liver lipids D. CCI4 hepatotoxicity potentiated by: 1. Microsomal enzyme inducers 2. Ethanol 1 V-~{y,44,\ 4 ., , 14 ko+.,noc 0 -~~a. Isopropyl alcohol~ 00 b. (c Acetone Diabetes w cN. co 0 0 . ,t c.. ~ 0 Kio
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a. Carbon tetrachloride b. Chloroform c. Tannic acid - treatment of burns, also added to barium enemate d. Phosphorus - yellow phosphorus, periportal liver injury, matches e. Tetracycline - esp. chlortetracycline, in pregnant women with kidney failure - fatty liver f. Ethanol 9. Aflatoxin (1) Food poisoning - seen especially in turkeys and other farm animals given peanut meal contaminated with the fungus Aspergillus flavin (2) Thought to play a role in the pathogenesis of cirrhosis and hepatoma in malnourished s • populations Locality Liver Cancer (Per 100,000 Persons/Year) Aflatoxin (ng/kg body wt/day) Kenya - High altitude 0.7 3.5 Thailand - Sangkhia 2.0 5.0 Kenya - Middle altitude 2.9 5.8 Kenya - Low altitude 4.2 10 CD Thailand - Ratburi 6.0 45 w Mozambique - Inhambane 25 222 c7-J CO h. Ethionine - fatty liver in females by decreasing ATP levels i. Orotic acid - fatty liver . K4
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(a) Hydrazine MAO inhibitors - including iproniazid, ~ isocarboxazid, nialamid, isoniazid and phenylzine. However, the non-hydrazine MAO inhibitor, tranylcypromine does not effect the liver. Iproniazid was introduced as a chemotherapeutic agent in the treatment of TB but was abandoned because it was more toxic than the equally effective compound, isoniazid. (b) Isoniazid (INH) (1) 1970 - 2,321 Capital Hill employees were found to have positive reaction to tuberculin and were begun in INH. During following 9 months, 19 had clinical signs of liver disease and 2 died from the illness. Hepatitis did not occur in 260 that declined INH therapy. (2) Hepatotoxicity more common in fast acetylators. Isoniazid3 Acetylisoniazid (3) Pharmacogenetics Slow: Eskimos Japanese Chinese . Fast: Egyptians Scandanavians Israelis Finns c. Halothane (1) Risk is 1 in 10,000 !L-~ Isonicatinic acid Acetylhydrazine ~ Reactive metabolites w • K6
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Curtis D. Klaassen • Dr. t0aassen is Professor of Pharmacology and Toxicology at the University of Kansas Medical Center in Kansas City, Kansas. He received his B.S. from Wartburg College in Waverly, Iowa In 1964, and an M.S. and Ph.D. in Pharmacology from the University of Iowa in 1866 and 1a68, respectively. He has been on the faculty at the University of Kansas Medical Center since 1968. Research Interests InGude the hepatobiliary disposition of xenobiotics, the toxicity of cadmium, and the hepatotoxicity of chemicals. Dr. Kiaassen is/has been an associate editor of a number of journals including the Journal of Pharmacology and Experimental Therapeutics and Toxicology and Applied Pharmacology. He has served on numerous national committees Including those with National Institutes of Health, Food and Drug Administration, National Library of Medicine, Environmental Protection Agency, National Academy of 8cionce and the National Toxicoiogy Program. He was president of the Society of Toxicology (USA) in 1990-1991, and presently is president of the International Union of Toxicology (IUTOX). Dr. baassen is author of the toxicology section, of Goodman and Gilman's Pharmacolopical Basic of Therapeufics and editor of Casarctl and Doull's Toxicology: The Basic Science of Poisons. CO w w CO - A ~ K CO
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0 Dimethylnitrosamine k. Thioacetamine - necrosis but little or no fat accumulation 2. Compounds acting to produce cholestasis a. C-17 alpha alkyl-substituted steroids (1) Methyltestosterone (2) Norethandrolone b. Lithocholic acid c. Alpha-naphthylisothiocyanate (ANIT) d. Manganese-bilirubin C. Properties of compounds which induce nonpredictable liver injury (some say by host idiosyncrasy or hypersensitivity!!) 1. The lesions cannot be reproduced in laboratory animals 2. Only a small fraction of exposed individuals are susceptible to this type of hepatic injury 3. Neither the occurrence of such lesions nor their severity can be correlated with the amount of drug administered (no dose response) 4. The appearance of the . lesions bears no constantt temporal relationship to the institution of drug therapy 5. Often the lesions are accompanied by extrahepatic manifestations of an allergic reaction such as fever, rash, arthralgia and eosinophilia D. Subclassifications of type II hepatotoxins 1. Compounds producing parenchymal injury (viral-like hepatitis) xs
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~ C. Diethyl maleate depletes GSH and increases bromobenzene hepatotoxicity D. 1,1-dichloroethylene (vinylidene chloride, saran wrap monomer) mechanism of hepatotoxicity is similar to bromobenzene in that it depletes glutathione and covalently binds to macromolecules E. Acetaminophen ACETAMINOPHEN HNCOCHj 0H P•!50 MIIED~iUNGON OXIDASE NCOCH] 1 OLUTATHtONE Toxic GLUCURONIDE INTERMEDIATE NUCLEOPHILIC CELL ~ MACROMOLECULES .~suti~l JHJTf'YM)MF CELL MACROMOLECULES ON f MERCAPTURIC ACtD Pathways of acetaminophen metaholism. CELL DEATH XI. EXAMPLE OF HEPATOTOXICITY BY TRAPPING AN ENDOGENOUS SUBSTRATE: GALACTOSAMINE i f K13
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Y ll. LOCALIZATION OF DAMAGE j~° A. Centrilobular damage (Zone 3) 1. Examples a. Carbon tetrachloride b. Bromobenzene c. Acetaminophen d. Most hepatotoxins ~ ~~ 2. Reasons a. P-450 b. Less oxygen--y Y° B. Periportal damage (Zone 1) - phosphorous, allyl alcohol and aflatoxin C. Midzonal damage (Zone 2) - beryllium ~ ' D. Massive necrosis KY MANIFESTATION OF DAMAGE A. Fatty liver - ethionine B. Necrosis - cell death - tannic acid and thioacetamide C. Both fatty liver and necrosis - carbon tetrachloride and -A ~~ ~~°~ chloroform ~ ~ e ~r ~ D. Cholestasis - chlorpromazine~~' E. Fibrosis and cirrhosis - chronic effect of repeated injury T"~'r~t ~~'~1 Q~C4 ~?~~ ~ ~ kya K2 0
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cytochrome P-450-catalyzed steroid hydroxylations by alpha-naphthoflavone. Mol. Pharmacol. 33, 493-499. Shou, M., Grogan, J., Mancewicz, J.A., Krausz, K.W., Gonzalez, F.J., Gelboin, H.V., and Korzekwa, K.R. (1994). Activation of CYP3A4:Evidence for the simultaneous binding of two substrates in a cytochrome P450 active site. Biochemistry 33, 6450-6455. Gallagher, E.P., Wienkers, L.C., Stapleton, P.L., Kunze, K.L., and Eaton, D.L. (1994). Role of human microsomal and human complementary DNA-expressed cytochromes P4501A2 and P4503A4 in the bioactivation of aflatoxin Bl. Cancer Res. 54, 101-108. Crespi, C.L., Penman, B.W., Gonzalez, F.J., Gelboin, H.V., Galvin, M., and Langenbach, R. (1993). Genetic toxicology using human cell lines expressing human P-450. Biochem. Soc. Trans. 21, 1023-1028. Idle, J.R., Armstrong, M., Boddy, A.V., Boustead, C., Cholerton, S., Cooper, J., Daly, A.K., Ellis, J., Gregory, W., and Hadidi, H. (1992). The pharmacogenetics of chemical carcinogenesis. Pharmacogenetics. 2, 246-258. Kadlubar, F.F. (1994). Biochemical individuality and its implications for drug and carcinogen metabolism: Recent insights from acetyltransferase and cytochrome P4501A2 phenotyping and genotyping in humans. Drug Metab. Rev. 26, 37-46. Relling, M.V., Lin, I.-S., Ayers, G.D., and Evans, W.E. (1992). Racial and gender differences in N-acetyltransferase, xanthine oxidase, and CYP1A2 activities. Clin. Pharmacol. Ther. 52, 643-658. Gonzalez, F.J. and Meyer, U.A. (1991). Molecular genetics of the debrisoquin-sparteine polymorphism. Clin. Pharmacol. Ther. 50, 233-238. Miles, J.S. and Wolf, C.R. (1991). Developments and perspectives on the role of cytochrome P450s in chemical carcinogenesis. Carcinogenesis 12, 2195-2199. Hanukoglu, I. (1992). Steroidogenic enzymes: Structure, function, and role in regulation of steroid hormone biosynthesis. J. Steroid Biochem. Mol. Biol. 43, 779-804. Osborne, M.P., Bradlow, H.L., Wong, G.Y.C., and Telang, N.T. (1993). Upregulation of estradiol C16cti-hydroxylation in human breast tissue: A potential biomarker of breast cancer risk. JNCI 85, 1917-1920. Nebert, D.W. (1993). Elevated estrogen 16a-hydroxylase activity: Is this a genotoxic or nongenotoxic biomarker in human breast cancer risk. JNCI 85, 1888-1891. i 0 Wolff, M.S., Toniolo, P.G., Lee, E.W., Rivera, M., and Dubin, N. (1993). Blood levels of organohclorine residues and risk of breast cancer. J. Natl. Cancer Inst. 85, 648-652. 0
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MENADIONE METABOLSM SCD 3 GSSG ~ GSSG xeductase CONJUGATES NADPH NADP K16 •
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1991;266:10019-10022. ~ - - -- • - - ---- 1991. Kalow W, Tang B-K. Use of caffeine metabolite ratios to explore CYP1A2 and xanthine oxidase activities, Clin. Pharmacol. Ther. 1991;50:508-519. 1991. Kalow W, Tang BK. Caffeine as a metabolite probe: exploration of the enzyme-inducing effect of ~ cigarette smoking , Clin. Pharmacol. Ther. 1991;49:4448. 1991. Nebert DW, Nelson DR, Coon MJ, Estabrook RW, Feyerseisen R, Fujii-Kuriyama Y, Gonzalez FJ, Guengerich FP, Gunsalus IC, Johnson EF, Loper JC, Sato R, Waterman MR, Waxman DJ. The P450 superfamily: update on new sequences, gene mapping, and recommended nomenclature. DNA Cell Bio1 1991-;10:1-14. 1991. Nebert, DW and Nelson DR. P450 gene nomenclature based on evolution In: Waterman, MR and Johnson, EF, eds. Cytochrome P450. Methods in enzymology. Volume 206. New York: Academic Press 1991:1-11. 1991. Porter TD, Coon MJ. Cytochrome P-450: multiplicity of isoforms, substrates, and catalytic and regulatory mechanisms. J Biol Chem 1991;266:13469-13472. 1991. White RE. The involvement of free radicals in the mechanisms of monooxygenases. Pharmacol Ther 1991;49:21-42. 992. Batt A-M, Siest G, Magdalou J, Galteau, M-M. Enzyme induction by drugs and toxins. Clin Chim Acta 1992;209:109-121. 1992. Coon MJ, Ding X, Pernecky SJ, Vaz ADN. Cytochrome P450: progress and predictions. FASEB J 1992;6:669-673. 1992. Fuhr U, Doehmer J, Battula N, Wolfel C, Kudla C, Keita Y, Staib AH. Biotransformation of caffeine and theophylline in mammalian cell lines genetically engineered for expression of single cytochrome P450 isofonns. Biochem Pharmacol 1992;43:225-235. 1992. Gonzalez FJ. Human cytochromes P450: problems and prospects. Trends Pharmacol Sci - 1992;3:346-352. 1992. Guengerich FP. Characterization of human cytochrome P450 enzymes. FASEB J 1992;6:745-748. 1992. Guengerich FP. Cytochrome P450: advances and prospects. FASEB J 1992; 6:667-668. 1992. Guengerich FP. Human cytochrome P-450 enzymes. Life Sciences 1992;50:1471-1478. 1992. Hollenberg PF. Mechanisms of cytochrome P450 and peroxidase-catalyzed xenobiotic ~ metabolism. FASEB J 1992;6:686-694. 1992. Kaminsky LS, Fasco MJ. Small intestinal cytochromes P450. Crit Rev Toxicol 1992;21:407- 422. 1992. Maurice M, Pichard L, Daujat M, Fabre I, Joyeux H, Domergue J, Maurel P. Effects of imidazole derivatives on cytochromes P450 from human hepatocytes in primary culture, FASEB J. 1992;6:752-758. 1992. McDonnell WM, Scheiman JM and Traber PG. Induction of cytochrome P4501A genes (CYPIA) by omeprazole in the human alimentary tract, Gastroenterology 1992;103:15091516. 1992. Proschaska HJ, Santamaria AB and Talalay P. Rapid detection of inducers of enzymes that protect against carcinogens, Proc Natl Acad Sci USA 1992;89:2394-2398. 1992. Rost KL, Brosicke H, Brokmoller J, Scheffler, Helge H, Roots I. Increase of cytochrome P4501A2 activity by omprazole: Evidence by the 13C-IN-3-methyll-caffeine breath test in poor and extensive metabolizers of S-mephenytoin, Clin. Pharmcol. Ther. 1992;52:170-180. human lung microsomai cytochrome P-4501A1 and its role in the oxidation of chemical carcinogens, Mol. Pharmacol. 1992;41:856-864. 1992. Wrighton SA, Stevens JC. The human hepatic cytochromes P450 involved in drug metabolism. Crit Rev Toxico11992;22:1-21. 1992. Zhang Y, Talalay P, Cho C-G and Posner GH. A major inducer of anticarcinogenic protective enzymes from bmccoli: isolation and elucidation of structure, Proc Natl Acad Sci USA 1992;89:2399- 2403. (7)
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~ HEPATOTOXICITY CURTIS D. KLAASSEN I. HISTOLOGY OF THE LIVER A. Light microscope po./c-Hepatic triad j I Central vein L,, B. The hepatic acinus • Thre hcpntirc nr.inus. the mir.rovnsr.ulnr unit of ivcr parenr.hymn. Thc acinir a.eis is (urmed hv the tcrmimtl hranchus of thre purt:d vr.mdc (TPV), the hr.palir. artcriulr. (IfAI and the 6ilc rluclulr. (f1U). llloud entr.rs thr acinar sinus- oids in zone I nnd flows sr.qur.ntiully throuph zone 2 and into nr.innr ronr, (1, where it exits via lhn terminal hr.puliro vnutdes ('rAV). "I'hre sinusuids n( znnc I nrn highly nnnslunmlir: while thuse of zone 3;ve straight and empty into the T!-IV in a radial arrangement. ! 4 µ ya /t ~ 1. portal vein ~ 2. hepatic arteriole 3. bile ductule- rm4n 4. lymphatic ~ 'y1 T c~f"~ Un'`,Lql~ni k sinusoid yr~' ~V °°+4'~e°t, bile canaliculi space of Disse ~ parenchymal cells (70% of number and 90% of mass) Kupffer cells ~ trilK~ q
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0 JiW- ;.in ~ 1N' %,; 1JL ~„y ~ ~-41 (2) More common with repeated exposure (3) In rats when microsomal enzymes induced (arochlor 1254 or phenobarbital) and low oxygen d. Cincophen - an antiarrhythmic agent. Occurred in only 1 in 1000, but mortality rate about 50% e. Zoxazolamine - a muscle relaxant and effective uricosuric agent f. Phenylbutazone g. Indomethacin h. Tricrynafen (Selacryn) (1) A uricosuric diuretic used in treatment of hypertension and heart failure (2) Marketed in May 1979 and recalled in Jan. 1980 because of 52 reports of liver injury with probable frequency of 1 in 5000. 2. Compounds producing cholestasis a. Chlorpromazine b: Carbutamide ~t{yY/ Ci. c. Chlorpropamide d. Oral contraceptives (1) Incidence is very low and this complication is also seen in late pregnancy (2) Most common in Scandinavia and Chile (3) Appears to be due to the estrogenic component because following recovery from K7
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9 H H H r I I ( * R- C= C- C- C= C- C- C= C- C- C= C- R ~ 1 f H ~H°[~ ~ H CHCl,~~i3((~ 'CC13 H H I I R- C= C- C- C= C- C- C= C- C- C= C- R H H Organic Free Radical H H H 1 1 I R- C= C- C- C- C= C- C= C- C- C= C- R H - r H 0 H 0 11 1 u - ~ H - C - C - C - H Organic Free H Radicals . ~e 7P malonic dialdehyde diene conjugation = 233 nm H H +0 2 H I I I R- C= C- C- C- C= C- C= C - C- C= C- R I I I H 0 H 0 I Peroxy Free Radical H H b ' H . I 1 l R- C= C- C- C- C= C- C= C- C- C= C- R 1 0 ~ H J\ H H / W W Organic Peroxide C~ (lipid hydroperoxide) ~J 4h. I;11
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• IMMUNOTOXICOLOGIC MECHANISMS RODNEY R. DIETERT
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A. Examples 1. Release from Mitochondria uncouplers, quinones, hydroperoxides, MPTP, Fe2+, Cda+ 2. Release from Endoplasmic Reticulum CCI„ bromobenzene, quinones, hydroperoxides, aldehydes 3. Influx through Plasma Membrane CCI4, CHCI31 dimethylnitrosamine, acetaminophen, TCDD 4. Inhibition of Efflux from the Cell cystamine, quinones, hydroperoxides, diquat, MPTP, vanadate B. Consequences of Disruption of Ca2+ Homeostasis 1. Alterations in Cytoskeleton a. Plasma membrane blebbing (1) Caa+ regulation of polymerization (2) Ca2+ -activated protease b. Alterations in plasma membrane channels 2. Activation of Phospholipases a. Ca2+ - and calmodulin-dependent b. Increased membrane permeability c. Stimulation of arachidonate metabolism K18 0
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• ~~Ua cFwl KIDNEY I. REVIEW OF RENAL PHYSIOLOGY ,) ?1 0 A. B. C. D. Renal blood flow = 1200 ml/min (25% of cardiac output) Renal plasma flow = 650 mI/niin Glomerular filtration rate = 120 mI/min (20% of plasma flow) Urine excreted is approximately 1 ml/min (99% is reabsorbed) WHERE FILTRATE IS ABSORBED E. Proximal tubule: 70% F. Ascending limb: 20% G. Distal tubule: 8-10% 11. FUNCTION OF KIDNEY A. Excretion of wastes ~ ~~ , Electrol te balance B y . C. Acid-base balance D. Erythropoietin ~ E. Vitamin D, to 1,25-dihydroxy-vitamin D$ II1. ASSESSMENT OF RENAL FUNCTION cc, ~ A. Urine volume B. Urinary pH , C~ ~ C. Sugar in urine c,rJ 0'~' -{S `.C.~ K19
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. XII. REDOX CYCLING AND OXYGEN RADICALS Qulnone Semlqulnone Hydroquinone NADPH -, . Oxyform of Scmiyuinone reductase ~ drug 0 NADP' ~ y Reduced forlnJ ~ Quinone drug of reduciase e e e e 02 --> 02 --~ H202 -> 0F-I• -> Hz0 REDOX CYCLING SuperoxidIe dismulase I GSH H202 I lase G55G Glutalh'One Ca -~peroxidase + 1110 + '011t0Yi' Purma riun and wctnbolism of U~ und 11,0, in hepalocyres. 00 W CXS CJ`I K15
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CH .,CH i D-GALACTOSE D-GLUCOSE OH NH2 D-GLACTOSAMINE D-GLUCOSAMINE AL ATOS,kiN1INE 'iVIETABOT:IS,NI PATIT4VA Y OROTATE -~ ~- UNIP -~- -~- UDP ~ -~ --)- UTP n V n V ATP ADP GALACTOSA-MINE GAL?.CTOSA-MIti'E-1-P gelactokinase ~ glucose galactose-l-purid,rletcansterase G`7 UDPG ~} ~`jglucose-l-P r UDP--1'epimerasc sr >,.r a~. Y.. UDP-GALACTOS.A.IINE~,,~ UDP-GLUCOSAINIINE ~ Decrease in 1) UDP-glucose 2) UTP 3) UDP K14 UDP-G1cNAc (acetylglucosamine) b UDP-Ga1NAc(acetylgalactosamine) Co W CN Cp 0 w ~ ~ i
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! XIII. CELL DEATH A. Two general types .1. Necrosis - Result of loss of cellular volume homeostasis 2. Apoptosis a. Programmed cell death b. Unlike necrotic cells, apoptotic cells show no evidence of increased membrane permeability XIV. CHEMICAL DESCRIPTION OF Ca++ HOMEOSTASIS Plasma membrane Endoptasmic reticulum Mitochondria Ca 2+ (10" 7M) CaZ+- bind ing proteins Caz+ ~ L~ Sy~ { 10-3 M) fxn--" Figure 1 Schematic illustration of intracellular Ca' compart- menlation. K17 W w cN w 0 v, ~ V
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i • 1. GSH conjugate 2. Gamma-glutamyl transpeptidase 3. Cysteinyl glycinase 4. C-S Iyase D. Therapeutic agents ~1Eror-oc*'z# 1. Analgesics a. Aspirin -- TD,,;zs/(,;,t~v 1r'it~o b. Phenacetin (1) Affect renal papillae r ~ ~ `Q`~ ~ c. Acetaminophen ,L, ~° ~~ (1) Decrease in GSH ~ Covalent binding In perfused kidney--covalent binding and decreased GSH '1~0,40 u 2. Anesthetics a. Methoxyflurane, biotransformed to oxalate and fluoride 3.- Antibiotics a. Aminoglycoside (1) Streptomycin, neomycin, kanamycin, gentamycin (2) Affect glomerular structure and function, but primary target is the proximal tubule b. Cephalosporin - type antibiotics . Cephaloridine Concentrates in kidney by active transport Probenecid blocks uptake and-toxicity Depletes GSH - May be biotransformed to reactive metabolite because phenobarbital increases toxicity Species selectivity correlates with GSH depletion x21 8338fl?51
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1982. Conney AH. Induction of microsomal enzymes by foreign chemicals and carcinogenesis by polycyclic aromatic hydrocarbons: GHA. Clowes memorial lecture. Canc Res 1982;42:4875-4917. 1982. Levin W, Wood AW, Chang RL, Ryan D, Thomas PE, Yagi H, Thakker DR, Vyas K, Boyd C, Chu S-Y, Conney AH, Jerina DM. Oxidative metabolism of polycyclic aromatic hydrocarbons toM ultimate carcinogens. Drug Metab Rev 1982;13:555-580. 1983. Nebert DW, Negishi M, Eisen HJ. Genetic differences in enzymes which metabolize drugs, chemical carcinogens, and other environmental pollutants. In: Tucker RE, Young AL, Gray AP, eds. Human and environmental risks of chlorinated dioxins and related compounds. New York:Plenum Publishing Corp 1983:441-462. 1984. Fujino T, West D, Park SS, Gelboin HV. Monoclonal antibody-directed phenotyping of cytochrome P450-dependent aryl hydrocarbon hydroxylase and 7-ethoxycoumarin deethylase in mammalian tissues. J. Biol. Chem. 1984;259:9044-9050. 1985. Anderson LM and Seetharam S. Protection against tumorigenesis by 3-methylcholanthrene in mice by p-naphthoflavone as a function of inducibility of inethylcholanthrene metabolism, Cancer Res 1985;45:6384-6389. 1985. Guengerich FP, Liebler DC. Enzymatic activation of chemicals to toxic metabolites. Crit Rev Toxicol 1985;14:259-307. 1985. Reik LM, Levin W, Ryan DE, Maines SL, Thomas PE. Monoclonal antibodies distinguish among isozymes of the cytochrome P-450b subfamily. Arch Biochem Biophys 1985;242:365-382. 1985. Thakker DR, Yagi H, Levin W, Wood AW, Conney AH, Jerina DM. Polycyclic aromatic hydrocarbons: metabolic activation to ultimate carcinogens. In: Anders MW, ed. Bioactivation of foreign compounds. Orlando, Florida:Academic Press, 1985:177-242. 1985. Watkins PB, Wrighton SA, Maurel P, Schuetz EG, Mendez-Picon G, Parker GA, Guzelian PS. Identification of an inducible form of cytochrome P-450 in human liver, Proc. Nail. Acad. Sci. U.S.A. 1985;82:6310-6314. 1986. Conney AH. Induction of microsomal cytochrome P-450 enzymes: The first Bernard B. Brodie lecture at Pennsylvania State University, Life Sciences 1986;39:2493-2518. 1986. Okey AB, Roberts EA, Harper PA and Denison MS. Induction of drug-metabolizing enzymes: Mechanisms snd consequences, Clinical Biochem 1986; 19:132-141. 1986. Ortiz de Montellano PR. Cytochrome P-450: structure, mechanism, and biochemistry. New• York: Plenum Press, 1986. 1986. Ortiz de Montellano PR. Oxygen activation and transfer. In: Ortiz de Montellano PR, ed. Cyctochrome P-450. New York: Plenum Press,1986:217-271 1987. Campbell ME, Grant DM, Inaba T, Kalow W. Biotransformation of caffeine, paraxanthine, theophylline, and theobromine by polycyclic aromatic hydrocarboninducible cytochrome(s) P-450 in human liver microsomes, Drug Metabl. Dispos. 1987;15:237-249. 1987. Campbell ME, Spielberg SP, Kalow W. A urinary metabolite ratio that reflects systemic caffeine clearance, Clin. Pharmacol. Ther. 1987;42:157-165. 1987. Guengerich FP. Mammalian cytochromes P450. Boca Raton: CRC Press, Vol.1,1987. 1987. Guengerich FP. Mammalian cytochromes P-450. Boca Raton: CRC Press, Vol. 2,1987. 1987. Nebert DW. P450 genes: structure, evolution, and regulation. Annu Rev Biochem 1987;56:945-993. 1988. Murrary GI, Barnes TS, Sewell HF, Ewen SW, Melvin WT and Burke MD. The immunochemical localisation and distribution of cytochmme P-450 in normal human hepatic and extrahepatic tissues with a monoclonal antibody to human cytochrome P-450. Br J Clin Pharmacol, 1988;25:465-475. 00 1988. Ziegler DM. Flavin-containing monooxygenases: catalytic mechanism and substrate w specificities. Drug Metab Rev 1988 ;9:1-32. C~j 1989. Andersson T, Regardth CG, Dahl-Puustinen ML, Bertilsson L. Slow omeprazole metabolizers are 0:~ also poor S-mephenytoin hydroxylators, Ther. Drug. Monitoring 1989;12:415-416. ° 1989. Guengerich FP. Characterization of human microsomal cytochrome P-450 enzymes. Annu o Rev Pharmacoi Toxicol 1989;29:241-264. ~ 1989. Guengerich, F. P. (1989) Polymorphism of cytochrome P-450 in humans. TIPS, 10, 107109 1989. Nebert DW, Jones JJ. Regulation of the mammalian cytochrome P1450 (CYP1A1) gene, Int. J. Biochem. 1989;21:243-252 1989. Nebert DW. The Ak locus: genetic differences in toxicity, cancer, mutation and birth defects, Crit. • Rev. ToxicoL 1989;20:153-174. 1989. Ortiz de Montellano PR, Stearns RA. Radical intermediates in the cytochrome P-450 catalyzed oxidation of aliphatic hydrocarbons. Drug Metab Rev 1989;20:183-191. 1989. Shimada T, Iwasaki M, Martin MV, Guengerich FP. Human liver microsomal cytochrome P450 enzymes involved in the bioactivatioh of procarcinogens detected by umu gene response in Salmonella
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X. EXAMPLE OF HEPATOTOXICITY PRODUCED BY COVALENT BINDING: BROMOBENZENE AND ACETAMINOPHEN H EPATOTOXICITY ~ A. Biotransformed by P-450 1. Toxicity potentiated by phenobarbital ' 2. SKF 525a or piperonyl butoxide decrease toxicity ~--- / B. Metabolic pathway ~ Covalently Microsomes . Nonenzymatic Bound to Macromolecules NADPH + oxygen ~o H Bromobenzene Epoxide , Epoxide Hydrolase S "i`"du~~ 5;a Transferase ' Nonenzymatic Rearrangement f (l , i o 3,4-dihydro- 3 4-dihydrox Y ydfVctif73 s yS~M µ 61~ :~ v5N 65 N ~ ~ , bromobenzene ~ Loss of Glycine and Glutamate A c tylate Mercapt~u`ric Acid ~ 0a ot~ 3,4-dihydroxy bromobenzene 0 i • xiz
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c. Tetracyclines ! (1) demeciocycline (2) outdated tetracyclines d. Amphotericin B E. Mycotoxins - a. Rubratoxin B b. Aflatoxin B1 c. Ochratoxin A (endemic Balkan nephropathy) d. Citrinin 0 K22
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Dietert - 4 VI. Mechanisms of Autoimmunity A. Factors Important in Autoimmunity B. Examples of Drug- and Chemical-Inducers of Autoimmunity VII. Mechanisms of Hypersensitivity A. Chemicals Important in Hypersensitivity B. The Antigen Pathway C. The Cytokine-Immunoglobulin Pathway VIII. Summary IX. References • 0
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Dietert - 9 . From: Davey, B. Immunology, Prentice Hall, 1990. As T cells differentiate within the thymus microenvironment, changes in gene expression enable functionally-distinct cell populations to arise. T lymphocytes can be immunoregulators or direct effector cells depending upon their phenotype. i
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Dietert - 3 0 IMMUNOTOXICOLOGIC MECHANISMS RODNEY R. DIETERT Institute for Comparative and Environmental Toxicology Department of Microbiology, Immunology, and Parasitology Cornell University OUTLIlVE . I. I. The Immune System as a Target Organ A. Cellular Components of the Immune System B. Soluble Immune Mediators C. Systemic vs. Regional Immunology D. Targeted Toxicity - AIDS E. Acquired vs. Innate Immunity Mechanistic Distinction in Immunotoxicology A. B. Direct Action of Chemicals on the Immune System Indirect Modulation of Immune Function III. Immunotoxicologic Evaluations A. B. The Balance of Practicality and Predictability The Tier System: Present and Future IV. The Role of Genotype in Immunotoxic Responses A. B. The Hypersusceptible Subpopulation Concept The Opportunities in Human Preventative Medicine V. Mechanisms of Immunosuppression A. The TCDD Story 00 0 B. C. D. Polycyclic Aromatic Hydrocarbons Benzene Toxicity Wood Preservatives: Tributyltin oxide w w w 0 E. Lead Toxicity w U, C-n
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rotein in urine • D E , , xcess p . E. Urine osmolality , ,(~ BUN C ` F . . G. Plasma creatinine l 1 ' H. Phenolsulfonphthalein (PSP) excretion ;~- I. PAH uptake by kidney slices z J. Histopathology 0 IV. SPECIFIC NEPHROTOXINS A. Metals 1. Mercury (mercuric chloride)-pars recta of the proximal tubule 2. Chromium (potassium dichromate)-proximal convoluted tubule 3. Cadmium (cadmium-metallothionein)-proximal tubule 4. Uranium-proximal tubule 5. Platinum (cisplatin)-proximal, distal and collecting ducts B. Halogenated Hydrocarbons 1. Examples a. Carbon tetrachloride C. 0 b. Chloroform (1) Enzyme induction increases toxicity (2) Depletes GSH <-(3) Species, sex and strain specificity (4) Biotransformed by kidney to pho` s ee c. Bromobenzene l" FO"'` G (1) Depletes GSH (2) Covalent binding (3) Proximal tubule necrosis 2. Mechanism ~ a. Proximal tubule b. Biotransformed to reactive compounds, with chloroform it appears that kidney biotransforms it to phosgene Hexachlorobutadiene K20 ,a a6l"A- ro,dic l ,1j) 83380350
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Dietert - 10 0 REGIONAL IMMUNOLOGY Regional Environmental Sites Specialized Cells Factors of Interest Lung Alveolar Macrophages Asbestos,',2,3 Gallium Arsenide4 Skin Langerhans Cells UVB Radiation,5 Pentamidine Isethionate,6 OxazoIone' Liver Kupffer Cells Ethanol$ G.I. Tract I.E. Lymphocyte r ' Dubois, C.M., et al., Amer. Rev. Resp. Dis. 139:1257, 1989. Roney, P.L., and A. Holian, Tox. App. Pharm. 100:132, 1989. 3 Nadeau, D. and D.A. Lane, Cell Biol. Toxicol. 4:13, 1988. 4 Sikorski et al., Fund. Appl. Toxicol. 13:843, 1989. 5 Streilein, J.W. and P.R. Bergstresser, Immunogenetics 27:252, 1988. 6 Rosenthal, G.L. et al., Tox. Appl. Pharm. 107:555, 1991. ' Ferreira, A.M., et al., J. Immunol. 127:2366, 1981. $ Eguchi et al., Hepatology 13:751, 1991. cxa Specialized sites within the body need host protection that is highly adapted to the w ~ features of that region. As a result, these sites are usually populated by novel ~ combinations of immune cells. Cs c.114 cN NJ
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Dietert - 7 CELLULAR COMPONENTS OF THE IMMUNE SYSTEM Thymus-derived lymphocytes - T cells Bursa-derived lymphocytes - B cells Natural Killer cells - NK cells Myelomonocytic cells - Macrophages Granulocytes Cellular components of the immune system arise from pluripotent stem cells through differentiation in distinct microenvironments. These cells cooperate in a coordinated fashion to provide host protection against disease challenges.
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Dietert - 6 I. THE IMMUNE SYSTEM AS A TARGET ORGAN 0 • From: Jerne, N.K., Sci. Amer. 229:52, 1973; and Hood et al. Irnmuno;)~, Benjamin Cummings, 1978. co w w ~ 0 ~ C"i Immune cells are widely dispersed in humans and other higher animals. As a ~ result, exposure to xenobiotics by virtually any route can result in some immune cell exposure.
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Dietert - 13 II. MECHANISTIC DISTINCTIONS IN IMMUNOTOXICOLOGY • DIRECT VERSUS INDIRECT IMMUNOTOXICITY Agent Primary Lesion Secondary Modulation Functional Change Chemical --> Chemical --> Thymic Lymphocyte ------> Thymic Epithelium -----> Altered T Cell Differentiation -----> Altered T Cell Differentiation -----> Function Altered T Cell Function Altered T Cell 0 Chemical --> Neuroendocrine or Hepatic Systems ---> Altered T Cell Differentiation ---> Altered T Cell Function Chemicals can exert a direct effect on developing or mature leukocytes. However, similar immune alterations can arise through xenobiotic modulation of non-immune target cells.
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Dietert - 15 THE HEPATIC-IMMUNE NETWORK' Casein -- Liver -'0' Altered Acute Phase Reactant Profile ~ Inhibition of Macrophage Elevated Serum Cytokir,e Production ""-- Amyloid A Protein ' Kaminski and Holsapple, J. Immunol. 139:1804, 1987. A likely mechanistic, pathway for casein-mediated immune suppression via altered hepatic gene regulation is illustrated. . 0 ~ V
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Dietert - 17 0 FUTURE IMMUNOTOXICOLOGY TESTING Individual Test I Percent* Paired Test Percen Plaque forming cells 78 NK activity 94 NK activity 69 Surface markers 90 Surface markers 83 Plaque forming cells 91 LPS response 50 Plaque forming cells 81 40 Delayed Hypersensitivity 57 Plaque forming cells 89 From: Luster et al., Fund. Appl. Toxicol., 18:200, 1992. * Percent concordance is indicated where greater than 20 analyses were reported. The results suggest that new test panels with a reduced number of endpoints may be feasible. 46
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Dietert - 8 r multipotent stem rUs from which all leukocytes ate derived T ceU precursors effector T cell maturing B cells effector B ceU T ceU depeadent B cd!-dependcnt (ceD-mediated) immuttitv (anubod)-med'tated) immtmity From: Davey, B. Immunology, Prentice Hall, 1990. CO The differentiation of T and B lymphocytes occurs in the thymus and bursa- CW i equivalent environments. The maturation of these cells in conjunction with ~ specialized epithelial cells is critical for the development of cell-mediated and ° w humoral immunity. ew to
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Dietert - 5 • IMMUNOTOXICOLOGIC MECHANISMS ABSTRACT The appropriate development and function of the immune system is a prerequisite of considerable importance to the health and livability of humans and animal wildlife. No other example emphasizing the necessity of immune surveillance and regulation is more striking than that of acquired immunodeficiency syndrome (AIDS). In this case, selective immune damage produces improper immune regulation which, in turn, results in immunologically-compromised individuals. While the relationship between immunocompetency and health is clear, predictive assessments of the impact of environmental-immune interactions presents ~ a particular challenge. This exists, in part, because the immune system is composed of multiple layers of host protective processes that are intertwined to ensure the integrity of the host. Furthermore, the immune system is dispersed within the host making the study of immunotoxicology strategically more complex than most investigations of single organ (e.g., kidney) toxicology. The immune system also has many communication loops with various organs (e.g., liver) and systems (neurological and endocrine). Therefore, the analysis of the immune system in isolation vs. an interactive immune system in vivo might offer different pictures of an environmental-host interaction. This presentation will illustrate present and probable future approaches to immunotoxicology testing. The importance of host genotype in immunotoxicology will also be discussed. Two specific examples of immunotoxic responses will be considered. Chemical structure-immune function relationships will also be discussed. In addition to immunosuppression, the capacity of environmental factors to contribute to autoimmunity and hypersensitivity will be described. Finally, two models will be presented illustrating novel opportunities for drug- or environmentally-induced immunomodulations. W w w a w um ___.3
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Dietert - 18 0 IV. THE ROLE OF GENOTYPE IN IMMUNOTOXIC RESPONSES GENETIC FACTORS AND TOXICITY Species Environ Factor Toxicity Control Rat Acetaminophen Kidney Strain-specific' Mouse Cyclophosphamide Immune system Strain-specific2 Drosophila Cadmium Lethality Strain-specific3 Japanese Aflatoxin-B, Lethality Selected Quail Trait" ~ Human Nickel Immune system HLA - D AS (Contact dermatitis) ' Tarloff et al., Toxicology 56:167, 1989. 2 Pevitsky et al., Int. J. Immunopharmacol. 7_:875, 1985. 3 Gill et al., Toxicology 56:315, 1989; and Christie et al., Biochem. Genet. 23:571, 1985. ° Marks and Wyatt, Science 206:1329, 1979. 5 Olerupa and Emtestam, Immunogenetics 28:310, 1988. cxy Genetic variation concerning the outcome of xenobiotic exposure is evident in aU.J broad phylogenetic spectrum of species including humans. ,~~-, ~ C) CD
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Dietert - 2 0 SPIE:A.iCEIi 131CiGRAPHICA.L TIVF()IZNiATION RODNEY R. DIETERT RECENT APPOINTMENTS Director, Institute for Comparative and Environmental Toxicology, Cornell University. Professor of Tmmunngenetics, Department of Microbiology and Immunology, Cotnell University. Adjuuct Professor, Depactrnent of Poultry Scicnec, North Carolina State University. EDUCATION T3.S., Duke University - 1974 Ph.D., The University of Texas at Austin - 1977 RECENT PROFESSIONAL ACTI'YITIES/HONORS Who's Who in Medicine and Health Care, ist Edition, 1997, Invited Keynote Speaker - 14th International Neurotoxicology Conference, 1996. Who's Who In America, 1995-present. Who's Who In American Education, 4th Edition, 1993. . Invited U.S. Congressional Testimony, 1995 Clean Water Act. Editor, Immunology Book Series, CRC Press, Inc. NTP-NIEHS Reviewer - Itnmunotoxicology Program NIEHS, Contract Panel member, 1988-95: AIDS Therapeutics, Chemical Immunotoxicology, Immunotoxicology of Xenobiotics and Dzugs. USDA - Animal Molecular Genetics Grants Panel (1992); Panel Manager (1993-1994) - Editorial Board Member, Animal Biotechnology Invited Speaker - 1991 SOT Course on Advanced Immunotoxicology 1995 Clyde Eby Memorial Lecturer - North Carolina State University, Raleigh, NC. 1995 YCTTAC Keynote Speaker - Istanbul, Tnrkey. International Biographic Center (England) - Advisory Board. RESEARCH PUBLICATIONS The speaker has over 200 publications in itrununotoxicology and immunogenetics. These include over fi() Peer-reviewed research papers published in 25 different journals. Additionally, the speaker has contributed toxicology opinion articles and public education information in l;opular mcdia such as the Now York Times, the'L.A. Times, the Chicago Th'lrane, Environmentat Update, and The Scientist. DDRFS.S: Institute for Comparative and Environmental Toxicology and Department of Microbiology and Immunology, Cornell University, C5135 Veterinary Mcd'znai Center, Ithaca, NY 14853 6401 Phone: (607) 253-4015 / FAX: (607) 253-3384 co _ E-mail: rrdlCOcornell edu w .
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Dietert - 12 • ACQUIRED VERSUS INNATE IMMUNITY Acquired Response Antigen Primed T Acquired Immune Antigen + Presenting Cell + Lymphocyte ----> Effector Function 0 Innate Respo-nse Natural Killer Cell Macrophage + Target ----> Innate Effector Function Granulocyte Acquired or adaptive immune responses are dependent upon the antigen driven clonal proliferation of T lymphocytes and sometimes B lymphocytes. As a result, acquired immunity may represent a race between microbe and/or tumor cell proliferation vs. immune cell clonal expansion. In contrast, innate responses do not require immune cells to proliferate but are dependent upon the recognition of a limited array of target structures on tumor or virally-infected cells. 00 w w ~ CO ~ ~ xa.
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Dietert - 14 i TWO EXAMPLES OF INDIRECT IMMUNOTOXICITY • Mouse Model TCDD -----> Impaired Thymic Epithelium -----> Improper T Cell Development' Casein -----> Altered Hepatic Regulation -----> Macrophage Suppression2 ' Greenlee et aL, Toxicol. Appl. Pharmacol. 79:112, 1985. Z Kaminski and Holsapple, J. Immunol. 139:1804, 1987. Two examples are provided in which environmental factors can produce profound immune alterations through non-immune primary cellular targets. cxs w ch+ co 0 CD W eN dN
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THE ALLOGENEIC PROBLEM One or a few genotypes must frequently serve as the prototype for an entire species. Dietert - 19 ! #
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Dietert - 26 0 Benzene BENZENE TOXICITYI Metabolism -------------> I Lang et al., Fund. Appl. Toxicol. Hydroquinone Intracellular SH Antiproliferation Reduced Macrophage Activation Benzene is an in vivo myelomonocytic toxin. Several metabolites appear to be immunotoxic to leukocytes. Hydroquinone inhibits lymphocyte proliferation by interacting with intracellular sulfhydryl groups. cxs w txi ~ w 0 UA ,,I co
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Dietert - 20 0 UVB IMMUNOSUPPRESSION' Mouse Strain % Ear Swelline Phenotype Balb/c 95 Resistant DBA/2 90 Resistant ~ C57B1/6 \ C3H/HEN 1 Wl'Ir 8 u~' ~~ ~10 Susceptible Susceptible - C, ~,'"~ ' Data from Streilein and Bergstresser, Immunogenetics 27:252, 1988. Mouse genetic background determines the capacity of ultraviolet B light to suppress contact hypersensitivity induced by DNFB. 0 CO W ~ C)IJ tp C) w ~ r")
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Dietert - 22 . THE XENOGENEIC DILEMMA Many animal species share extensive similarities in xenobiotic metabolism and in general immune characteristics. However, most species are not identical in gene composition and/or structure relative to these two considerations. As a result, species differences do exist in environmental-immune interactions. Co w i `14 CO CD Chl ~
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Dietert - 21 • IMMUNOTOXICOLOGY TESTING C57B1/6 X C3H B6C3F, •
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Dietert - 24 • MOUSE TCDD EFFECTSI • Perinatal Adult Thymic epithelium Terminal B cell maturation T cell maturation Complement factors T cell regulation ' Information from Luster et al., J. Immunol. 140:928, 1988; Greenlee et al., Toxicol. Appl. Pharmacol. 79:112, 1985; Holsapple et aL, J. Pharmacol. Exp. Ther=: 2 1:518, 1985; White et al., Toxicol. Appl. Pharm. 84:209, 1986. TCDD exhibits a targeted effect on the mouse immune system. The effect has both indirect and direct components. Developmental status is apparently important in the influence of TCDD on the immune system. co cN w ~ 00 0 w V 01\
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Dietert - 16 0 III. IMMUNOTOXICOLOGICAL EVALUATIONS PANEL FOR DETECTING IMMUNE ALTERATIONS FOt3.OwING CHEMICAL OR DRUG EXPOSuRE IN RODENTS' Parameter SCREEN (Tier I) Immunopathology 0 Humoral-mediated immunity Cell-mediated immunity AnnvE+*t= Nor&pecific immunity COMPREHENSIVE (Tier lI) Immunopathology Humoral-mediated immunity Cell-mediated immunity Nonspecific immunity : Host resistance challenge models(endpoints)° i~ ~ro~ ('i7 u . a Procedures Hematology-Complete blood count and differential Weights-Body, spicen, thymus, kidney, liver Cellularity-Spleen Histology-Spleen, thymus, lymph node Enumerate IgM antibody plaque-forming cells to Tdependent antigen (SRBC). LPS mitogen nspoaso Lymphocyte blastogenesis to mitogens (Con A) and mixed leukocyte response against allogeneie leukocytes (MLR) Natural killer (NK) cell activity Quantitation of splenic B and T cell numbers Enumeration ofIgG antibody response to SRBCs Cytotoxic T lymphocyte (C1L) cytolysis. Delayed hypersensitivity response (DHR) Macrophage funetiontluantitation ofnesident peritoneal cells, and phagocytic ability (basal and activated by MAF) Syngeneic tumor cells PYB6 sarcoma (tumor incidence) B 16F10 melanoma (lung burden) Bacterial models L'uteria monoc}yogertes (mortality) Streptococcus species (mortality) Viral models inQuena(moriality) Parasite models P)armodimn ynelii (Parasitemia) ' Tbe testing panel was developed usiug B6C3FI fanak mice. ' For any particular chemid tested only two orthree hou resistancc modelsare selected forexaminatioa ~ From: Luster et al., Fund. Appl. Toxicol. 1G:2, 1988. The current tier system of assays for immunotoxicology testing as endorsed by the National Toxicology Program (NTP) is shown.
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Dietert - 27 TRIBUTYLTIN IMNIUNOTOXICITYl TBTO -----> Rats ----- > Thymic Involution Thymocyte Apoptosis Immune Parameters: Altered Antibody Responses Reduced DTH Responses Reduced Natural Killer Cell Activity Reduced Bacterial Clearance ' Smialowicz et al., Toxicology 64:169, 1990; Verdier et al., J. Toxicol. Env. Health 32:307, 1991; Raffray, M. and G.M. Cohen, Arch. Toxicol. 65:135, 1991. 0 0 Tributyltine compounds target thymus but the actual immunotoxic route remains to be determined. Apoptosis may be one cellular response.
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Dietert - 23 V. MECHANISMS OF IMMUNOSUPPRESSION Table 1. Examples of Immunologic Changes Associated with Xenobiotic Exposure Class Polyhalogenated aromatic hydrocarbons Metals Aromatic hydrocarbons (solvents) Polycyclic aromatic hydro- carbons Pesticides Organotins Aromatic amines Oxidant gases Particles Natural products Abused drugs Therapeutics Others Exampte' TCDD, PBB, PCDF, PCS Lead, cadmium, arsenic, methyl mer- cury Benzene, toluene, hexachlorobenzene DMBA, BaP. MCA Tdmethyt phopshorothioate, carbofu- ran, chlordane TBTO Benzidine, acetyf aminoftuorene NO,, 0,, SO, Silica, asbestos Selected vitamins, antibiotics, fungal products, vinca alkaloids, estrogen, plant alkaloids Ethanol cannabinoids, cocaine. opioids Diphenylhydantoin, lithium Nitrosamine, BHA TCDD = 2,3,7,8-tetrachlorodibenzo-p-dioxin; PBB = potybrominated biphenyls; PCDF = pctychlorinated dibenzofurans; PCB = polychlorinated biphenyls; DMBA = dimethytbenzanthracene; BaP = benzo(a)pyrene; MCA = methylcholanthrene: TBTO = bix(lri-n-butyltin)oxided; BHA = butyiated hydroxyanisole. From: Luster et al. Ann. Allergy 64:427, 1990. v 011 • • 0 Categories of xenobiotics known to produce immunotoxic changes are illustrated.
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NOMENCLATURE FOR THE ! IOCLEAVAGE OF CARBINOLAMIDE CYTOCHROME P450 ENZYMES (1~J H,H 0 Ci H/ •.H li~~ N~\C 1 89 FATE OF METABOLICALLY ACTIVATED CYCLOPHOSPHAMIDE Table 1. Ihwnt navkw and IMl.lad Areclsa un the Pjlochromo P-r5ow ACevaeon of mubyene by hlwnan cyfochronra P45e.niyrm.. (1990) nMcMrlenr ol cylocMame P-M uW)oia. (199a) Tha 19ee BrrrN B. Brod6 Award tacbwe /unceonal dlwxNty of Mpaec rytachromea P45o. (1990) B.NOevlty In the Inhibinm of memmalbn cylochromes P4ea by dnmlcal agenb- (199a) EvokNan o/ tlr P450 p.ne nperfamity: animelylant'mdan', moleculv ddve and human genetk an.nncea In dmy oxklatlon. (1troo) EnrynrMduceon In dr tytnchrome P-45a ryslem. (1990) Wnae polymorpHems of dmp metebollam (1990) Ch.nelolindon ol rotee of huwwn cytodwome P45a enrymae In earUnopan metabollam (199U) O.)dtllon of taldc and tarcMopelYc dwrticrla by human cytochrome P450 wntyoew (1901) Maceoru ud wi9nlficance of eylocMome P-460.n:ylna (1991) CnwpnSoN tLrg InMShcon st1M.s. (1991) OavNOpawnb and pnpadvn an 1M role af eylnehome Plsaa In errnicY earelnop.rrsl.. (1991) Plsa wnd hunan eanear. (1991) cDNAaapwaadhurn.ncytacLrorneM50a: enwwep.olmoMn:IrlodwlopYmdhurtxnd.k.awamant (1992) CylMiwaww P450 MlepolGly ol holomw, nlbelMw, nd catlyee and rwyul.tary machanlaon. (1991) ThelMSlrenhapatlecytodeomeeP450Involvedlndruynl.tdwa.rn (1992) P45DOnxynloe,MhlbleonmrcMnfam.,yrrw8engulaeonandaM.cleofavoraw..e. (1992) 4ylotlepM. P-/50111 np: .ituelulN, fllnctlom, proprtlN and relevant hun.n fomY. (1992) f.Ytodeom. P45(teem:aypaaography, oxygen acavaaon,.nd Nacbon Yanhr. (1999) Oyludrome P45a: edvaneee and preapwcta (1992) Odd.tivW and reductWe nlMeboll.nl by eyloclwolna. (1992) kNNrnlonwi of cyloWwanl. P45D and pr:rmddatecataynd lwnoblosc mot.boN.m. (1992) CYtachromw P450: progreaa uid preaceam. (1992) Ciwedadi.eon of human cytochrom. P450 wraym... (to92) Small lma.tinal cyloclwomwa-P45a. (19G2) IWrrn cytochroma P-450.nzymea. (192) 11um.n cytoclwomee PISa: problMna and pro.p.cla (1962) 82L8 (1) Enzymes with less than 40% amino acld sequenc. Identify are assigned to different gene families (1,2,3, etc.). (2) Enzymes that are 40 to 55% Identical are clessified s members of different subfamllles (2A, 2B, 2C, elc.). (3) Enzymes that are more than 55%Identlcel are classffled as members of Ihe same subfamily (2A7, 2A2, etc.). (4) Each designation is prefaced by "CYP" and the name Is Italicized when referring to the gene. For example, the gene CYPIA2 encodes for the enzyme CYP1A2. Prolotlc.nt.u,A e«Nnu9wi... P,IndpJ F,ab.raw po.ct..nun q namaua tr,.n nx rnryn~ Pa501a1 PISe]Et ~ ,(e~~±..MCT.P~cykb ~¢aN/.nylmn. C.Aw,1..arl~lulE. PlW 1.2 Chlwcbm zir,..M.mn.)wa.o. ww~N.nemiwn. 2MdnoAUaw~. TW/no,IhyNn. ?MtlnwnM.en. E\y4n.Oblak. .nyw. dem.td. ?NM,qa.BdmMpkners~..y 1.Satl,bqwnP.n. tqAmulM ?Amlrp3.l.Eflm.erytndd.ml[4,S ~mmIM1. AqWm..rn. Wrylbunn. acp,nroiw qimie.ux. EtM..b.,H. >~InPt,~,.nN5/fyyaaq..~M„e,y vsyiub..." xwmnmlpydea(l.z-.a.z-qime..a. ,wo.mn.p,., ?N.pheyl.min. aw.nne,po.~N Psom. aw,a~at.m.nNe#~w~Mnw..q.s nn.b.nei blunlnqknn aY~„ ej 541pm.EryW, PMOtwe 6.mam~rw MNewoAee mb. lFaRr,'O~ory-T.9GM1yaaE.nc{.~q.m N 9,iPneyJrarO,ltr UM1)drtb.rvc(G}Iw.nhm. ],Fqnyaory-]..dnryo-o].1? nmMN~W.nn,..n. Tmt2smvomy.opyp po.Pn.t. lretp:.w. SOME CHARACTERISTICS OF CYP1A1 (1) Catalyzes the bloactivatlon of polycyclic aromatic hydroarbons and arylemines. (2) Is not constitulively expressed In the liver of laboratory animals and humans (3) Is Induced by cigarette smoke (extrahepalic), polycycllc aromatic hydrocarbons, TCDD, Vnephthoflavone and food additives. (4) Intluctlon Is medlated by a cytosolic receptor proleln, the AMreceptor. Iat 1. tnnihitnA Lv .. nnnMl.nlm.n.n" 5.,, n•.. o~.~R.m"~
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Dietert - 25 MODULATION OF ANTIGEN PRESENTATION BY 0 POLYCYCLIC AROMATIC HYDROCARBONS Compound Macrophage Parameter Benzo[a]pyrene -Methycholantrene 7,12 Dimethybenz[a]anthracene Increased IL-1 Production after in vitro Exposure,' Altered Antigen Uptake2 Altered Class II Expressionz Decreased Antigen Presentation Capabilities3 Decreased Antigen Presenting 0 Capabilities ' Lyte, M. and P.H. Bick, Int. J. Immunopharm. 8_:377, 1986. 2 Myers, M.J., L.B. Schook, and P.H. Bick, J. Pharm. Exp. Ther. 242:399, 1987. 3 Johnson, B.E., R.G. Bell, and R.R. Dietert, Immunopharm. Immunotox. 12:237, 1990. ° Yamashita, U. and T. Hamaoka, Gann 73:773, 1982. Three widely studied polycyclic aromatic hydrocarbons have apparent similarities in their targeted effect on the immune system. !
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Dietert - 35 • IMMUNOGLOBULIN SWITCHING PATHWAY IgM B-lymphocyte IgGI B-lymphocyte IgE B-lymphocyte i Interleukin-4 + LPS Specific cytokines influence the heavy chain switch among populations of B-lymphocytes. High levels of interleukin-4 favor IgE production. w.a
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Dietert - 32 0 VII. MECHANISMS OF HYPERSENSITIVITY PROBABLE INDUSTRIAL ALLERGENS' Compounds Platinum Salts Formaldehyde Grains Cotton Dust ` Organophosphate Insecticides • Polyvinyl Chloride Products Vegetable Gums Diisbcyanates i Antibiotic Dust, Piperazine, Ampicillin Wood Dust Exposure Metal Refining Fabrics, Laboratories Farmers, Mill Operators, Bakers Textiles Farmers Meat Wrappers Printers Chemical Industry Pharmaceutical Carpenters, Mills ' Modified from Dean, J.H., M.I. Luster, A.E. Munson, et al. Immunotoxicology and Immunopharmacology, Raven Press, New York, 1985.
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Dietert - 29 DIFFERENTIAL EFFECTS OF LEAD ON T CELL FUNCTION A Hypothesis ~~,,w`~A Hypothesis TH, Activity ~ Reduced Cell-mediated Immunity • ~ TH2 Activity + Within T cell function, lead appears to alter the balance of Th, vs. Th, activity. 0
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Dietert - 31 EXAMPLES OF DRUG- AND CHEMICAL-INDUCED AUTOIMMUNITY Druas Diseases Penicillamine Systemic Lupus Erythematosus' Myasthenia gravis'- Myositis3 Sclerosis^ Hydralazine Systemic Lupus Erythematosus5 Environmental Chemicals Mercuric chloride Autoimmune nephritisb (in rats and other species) Rapeseed oil (TOS) Autoimmune scleroderma' ' Harpey et al., Lancet 1:292, 1971. Z Buchnall et al., Brit. J. Med. 1:600, 1973. 3 Halla et al., Amer. J. Med. 77:719, 1984. 4 Nishikai et al., Arch. Dermatol. 110:253, 1974. 5 Russell et al., Quat. J. Med. 65#246:845, 1987. 6 Aten et al., Amer. J. Pathol. 133:127, 1988. ' KammulIer et al., J. Toxicol. Clin. Toxicol. 26:157, 1988. Certain drugs and chemicals have a well-known association with specific autoimmune conditions. In the last example, rapeseed oil is only a suspected causative agent. • _ r
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Dietert - 37 IX. REFERENCES . Aten et al., Amer. J. Pathol. 133:127, 1988. Balkwill, F.R. and F. Burke, Immunol. Today 10:299, 1989. Christie et al. Biochem. Genet. 23:571, 1985. Davey, B. Immunology. A Foundation Text, Prentice Hall, Englewood Cliffs, NJ, 1990. Dean, J.H., M.I. Luster, A.E. Munson, et al. Immunotoxicology and Immunopharmacologv, Raven Press, New York, 1985. Decker et al. J. Leuk. Biol. 45:139, 1989. Dubois, C.M., E. Bissonnette, and M. Rola-Pleszczyiski, Amer. Rev. Resp. Dis. 139:1257, 1989. Eguchi, H., P.A. McCuskey, and R.S. McCuskey, Hepatology 13:751, 1991. Ferreira, A.M., M. Hurme, M. Kaartinen et al., J. Immunol. 127:2366, 1981. Gill et al. Toxicology 56:315, 1989. Greenlee, W.F., K.M. Dorf, and R.D. Iron, Toxicol. Appl. Pharmacol. 79:112, 1985. Blalla, J.T., F. Fullahi, and W.J. Koopman, Amer. J. Med. 77:719, 1984. Harpey, J., B. Collie, R. Moulias, J. Goust, Lancet 1:292, 1971. Holsapple, M.P., D. McNerney, D. Barnes, and K. White, J. Pharmacol. Exp. Ther. 231:518, 1985 Hood, L.E., I.L. Weissman, and W.B. Wood, Immunoloev, 00 Benjamin/Cummings Pubi. Co., Menlo Park, CA, pp. 467, 1978. ~ ~ 00 Jerne, N.K., Sci. Amer. 229:52, 1973 ~ ~,,, co 11tr
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Dietert - 28 LEAD IMMUNOTOXICITYI ~ i~ Y /7 u~u/ -r' ;/; . Reduced Cell-Mediated Immunity , p Pb ----> Macrophage i Reduced Antigen-Priming of T Lymphocytes ' Recently reviewed in: Zelikoff et al., Fund. Appl. Toxicol. 22:1, 1994. Lead alters macrophage function reduced both macrophage innate immune responses and the antigen-priming of certain T lymphocytes. 00 CM C,J ~ CO C7 Lj_j t:3 0
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Dietert - 36 • 1. 2. 3. 4. 5. ~ 6. 7. 0 VIII. SUMMARY Significant immune suppression or altered immune regulation can result in significant health risk to the host. Environmental factors producing immunotoxicity can operate via direct or indirect pathways in modulating the immune system. The impetus in immunotoxicology evaluations is to move toward fewer endpoints with a retention of high reliability. In this way, more compounds can be evaluated. At the same time, there is a desire to broaden the genotypic base of immunotoxicology data to better ensure the reliability of xenogeneic extrapolations to humans. `T s V .-~~) It is clear that compounds can exert either broad or narrow influences on the immune system. The profile of the influence can change with development and be under genetic control. While a diversity of mechanisms exists, certain classes of compounds appear to share mechanisms in attacking the immune system. This suggests that it will be possible to enhance preventative medicine through knowledge of chemical structure-hostimmune function relationships, and the influence of human genotype in mechanistic processes. Cb
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WS2 i Mechanisms of Neurotoxicity William Slikker, Jr:, Ph.D. I. Overview A. B. C. Definition of Neurotoxicity Occurrence/Incidence Human Health and Economic Costs II. Assessment of Neurotoxicity A. B. C. D. Neurochemistry Neurophysiology Neuropathology Behavior III. Postulated Mechanisms of Neurotoxicity A. Excitotoxic Hypothesis 1. Glutamate 2. Domoic Acid/Kainic Acid B. Oxidative Stress 1. Peroxides 2. Metals C. Mitochondrial Dysfunction . 1. MPTP 2. Dideoxynucleosides IV. Conclusions co W Cnl i Ot C) ~ 4~-
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MECHANISMS OF NEUROTOXICITY
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i WS 1-1 William Slikker, Jr. 0 i Current Professional Activities: Director, Division of Neurotoxicology, National Center for Toxicological Research, FDA Co-Author of Principles of Neurotoxicity Risk Assessment and Neurotoxicology: Approaches and Methods Adjunct Professor, Department of Pharmacology and Interdisciplinary Toxicology and Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, Arkansas Associate Editor: NeuroToxicology Councilor, Teratology Society Past-President, Neurotoxicology Specialty Section, Society of Toxicology Member, SOT Task Force to Improve the Scientific Basis of Risk Assessment Background: B.A., University of California, Santa Barbara, California; M.A., University of California, Santa Barbara, California; Biological Sciences (Biochemistry and Endocrinology); Ph.D., University of California, Davis, California; Pharmacology and Toxicology. Postdoctoral Training, Drug Research and Evaluation Program and Perinatal Program, NCTR; Institute of Toxicology and Embryopharmacology, Free University, Berlin Publications- Approximately 185 peer reviewed research publications and contributions to proceeding publications, books, and review journals.
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0 PROTECTION AGAINST.. FREE RADICAL MEDiATED. TISSUE INJURT o MARY TREINEN MOSLEN ~.
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/~'K r-s 1989. Shimada T, Martin MV, Pruess-Schwartz D, lblarnett IJ, Guengerich FP. Roles of individual human cytochrome P-450 enzymes in the bioactivation of benzo(a)pyrene, 7,8-Dihydroxy-7,8- dihydro€aenzo{a}pyrene, and other dihydrodiol derivatives of polycyclic aromatic hydrocarbons. Cancer Res 1989;49:6304-6312. *1940. Adamson RH, Snyderwine EG, Thorgeirsson UP, Schut HAJ, Turesky R.T, Thorgeirsson SS, Takayama S, Sugimura T. Kato R, Yamazoe Y, Yasumori t. Pharmacogenetics and polymorphism of human P-450 which activates and detoxicates xenabifltics and carcinogens. In: Ernster L, Esumi H, Jujii Y, Gelboin liV, Kato R, Sugimura T, eds. Xenobiotics and cancer, Proceeding of the 21st international symposium of the Princess Takamatsu cancer research fund. Tokyo: Japan Scientific Societies Press 199Q:288-3f}2. 1990. Andersson T, Cederberg C. Edvardsson Ci, Heggelund A, Lundborg P. Effect of omeprazole treatment on diazepam plasma levels in slow versus normal rapid metabolizers of omeprazole. Clin Pharmacol Ther 1990;47:79-85. 1990. Berthou F, Flinois TP, Ratanasavanh D, Beaune P, Riche C, GuilIouzo A. Evidence for the involvement of several cytochromes P-450 in the first steps of caffeine metabolism by human liver microsomes, Drug Metabl. Dispos. 1990;19:561-567. 1990. Diaz D, Fabre i, Daujat M, Saint Aubert B, Bories P, Michel H, Maurel P. Omeprazole is an aryl hydrocarbon-like inducer of human hepatic cytochrome P450, Gastroenterology 1990;99:737-747. 1990. Gonzalez FJ, Nebert DW. Evolution of the P450 superfamily: animal-plant 'warfare', molecular drive and human genetic differences in drug oxidation. Trend Genet 1990;6:182186. 1990. GGonzalez FJ. Molecular genetics of the P450 superfamily. Pharmacol Ther 1990;45:1-38. 1990. Guengerich FP, MacDonald TL. Mechanisms of cytochrome P-450 catalysis, FASEB I 1990;4:2453-2459. 1990. Heim M and Meyer UA Genatyping of poor metabolizers of debrisoquine by allelespecific PCR amplificaiton. Lancet 1990;336:529-532. 1990. Levin W. The 1988 Bernard B. Brodie Award Lecture functional diversity of hepatic cytochromes P-45It. Drug Met Dispos 1994;18:824-83U. 1990. McLemore GL, Adelberg S, Liu MC, McMahon NA, Yu SI, Hubbard WC, Czerwinski M, Wood TG, Storeng R, Lubet RA, Egglesto 7C, Boyd MR, Hines RN. Expression of CYPIA3 gene in patients ~with lung cancer- evidence for cigarette smoke-induced gene expression in normal lung tissue and for pulmonary carcinomas. I Natt Cancer Inst 1990;82:1333-1339. 1990. Meyer UA. Molecular genetics and the future of pharmacolgenetics. Pharmacol Ther 1990;46:349-355. 1990. Murray M, Reidy GF. Selectivity in the inhibition of mammalian cytochromes P-45U by chemical agents. Pharmacol Rev 199il;42:85-1fl1. 1990. fOkey AB. Enzyme induction in the cytochmme P450 system. Pharmacol Thet 1990;45:241-298. 1990. Pichard L, Fabre 1, Fabre G, Domergue J, Saint-Aubert B, Mourad G and Maurei P. Cyclosporin A drug interactions: Screening for indicers and inhbitors of cytochrome P-450 (cyclosporin A oxidase) in primary cultures of human hepatocytes and in liver microsomes. Drug Metab Dispos, 1990;18:595-G0b. 1990. Roberts EA, Johnson KC, Harper PA, Okey AB. Characterization of the Ah receptor mediating aryl hydrocarbon hydroxylase induction in the human liver cell line Hep G2, Arch. Biochem. Biophys. 1990;276:442-450. 090. Tefre T, Borresen A-L, Ryherg D, Haugen A and Brogger A. Allele association studies of the P45©FAland P45!?IIDl DNA polymorphisms in lung cnacer patients and controls. In: Drug Metabolizing Enzymes: Genetics, Regulation and Toxicity, Proceedings of the Eighth International Symposium on Microsomes and Drug Oxidations (Ingelman-Sundberg M, Gustaffson J-A and Orrenius S, Eds). Karolinska Institute, Stockhuha,199!}:pp51. 1990. Vang 0, Jensen B and Autrup Herman. Induction of cytochrome P450IA1 in rat colon and liver by iadole-3-carbirrol and 5,6-benzoflavone, Caminogenesis 1990; i 1:1259-1243. 1991. Andersson T. Bergstrand R, Cederberg C, Eriksson S, Lagerstrom P-G and Skanberg I. Omeprazole treatment does not affect the metabolsim of caffeine, Gastroenterology 1991;10I:943-947. ~ 1991. Andersson T. t3meprazoIe drug interaction studies. Clin Pharmacokinet 1991;21:195-212 1991. Danjat M, Pichard L, Fabre I, Pineau'T, Fabre G, Bonfil C and Maurel P. Induction protocols for ~ cytochmme P45{?IIIPi in vivo and in primary cultures of animal and human hepatocytes. Meth Enzmol ~ 1991;206:345-353. 1991. Gleizes C, Ecckhoutte C, Pineau T, Alvinerie M, Galtier P. Inducing effects of oxfendazole on ~ cytochmme P4501A2 in rabbit liver, Biochem. Phannacol.1991;41:1813-1820. p +1991.Gonzalez FJ: Crespi CL, Gelboin HV. eDNA-expressed human cytochrome P450s: a new age :o of molecular toxicology and human risk assessment. Mutat Res 1991;247:113127. 199i. Guengerich FP. Oxidation of toxie and carcinogenic chemicals by human cytochrome P-45Q
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TYPE I OPIDN Other OrOanophosphorus Esters C 0 1( O-P-O TPP 0 CH, ~ r"\J CH, i CHa O 1) O-P-O I TOCP TMCP + O CH, r \O-P-O-[~ ~}C 11 CH, CH, I CH, TPCP TPCPi Figure 2 Structures of some Type I and Type ll compounds. Threshold Single Dose for Induction of Type I and II OPIDN in Hens Type I (pentavalent organophosphorus Dose esters) (mg/kg) Route TOCP 62.5 s-c° TOCP 250 Oral' DFP 0.25 s.c.' Cyanofcnphos 5 Oral` EPN 25 Oral` Leptophos 100 Orah EPDP 800 Oral" DEF 100 Orald DEF 2S0 Dermald Type II Dose (Organophosphites) (mp/kg) Route TPPi 250 TOCP; 1919 ' Carrington and Abou-Donia (1966a). b Smith et aL, (1932). `Abou-Donia (1979). d Abou-Donia er af., (1979b). TYPE II OPIDN Orparnophosphttes TPPI CH O-P-O p CHs C TOCPI H3 TMCPi CH, + + + + WS13 9 w
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1 v(V~IGlJ Page 2 . • Protection Against Free Radical Damage I. Types of Protection Against Free Radicals A. Detoxification of Reactive Oxygen Species (ROS) B. Control of ROS Formation by Phagocytes II. Control of Lipid Peroxidation A. Detoxification of the Products of Lipid Peroxidation B. Consequences of Inadequate Control 1. Keshan's Disease 2. Deficiency of Glucose 6-P Dehydrogenase III. Distribution of Protective Systems Within Cells and Tissues IV. Adaptive and Therapeutic Protection V. Systemic Control of Fe and Cu A. Regulation of Fe and Cu Homeostasis B. Consequences of Excess Fe or Cu 1. Hemochromatosis 2. Wilson's Diseases cxo (N Uj co c~3 ~
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~ ~ 100% 100 U X 0 2 ~ ~ z a 80, a ~ 0 c 60 , tJ ~ a 46% 2 a N ~ 40. ~ 0 a E 0 U 0 20. e ~O ~ Type I Type II (Other (Organophosphites) Organophosphorus Compounds) Figure 1 Percentage of Type I and Type 11 compounds producing OPIDN. 0 0 0 II II II R4P-O-R R-0-P-0-R R-O-P-O- OH F 0 0 R I I I I HT6 DFP Phoephorylated "Aged" RTE HTE Figurc 7 Binding and aging of OP compounds to NCE. WS14
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Page y 4 i How Do Antioxidants Scavenge Free Radicais ? Reaction of Vitamin C (Ascorbate) + RH L-Ascorbic Acid Ascorbyl radical Reaction of Vitamin E (a-Tocopherol) CHj CH3 HO OiO i CH3 a-Tocopherol Dehydro-L- ascorbic acid I* r CH CH, CH^(~ ~7/~'HO-- ~~- 3 1 1 1 CI6H31 CH, a-Tocopheroiquinone + RH
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Neurotoxicity may be defined as any adverse effect on the structure or function of the central and/or peripheral nervous system by a bioiogical, chemical, or physical agent Neurotoxic effects may be permanent or reversible, produced by neuropharmacofogicai or neurodegenerative properties of a neurotoxicant, or the result of direct or Indirect actions on the nervous system. Many of the relevant effects can be measured din'ectty by neurochemicai, neurophysiological or neuropathoiogical techniques, whereas, others must be Inferred from observed behavior. 96209228 Neurotoxicology Program Social Impact • One out of four /lmericons will wffer from a brain-reiated disorder at some point In fh•ir,if•. • One out of ten school age children have a funcAonal deficit. NCI'R/I'oA Neurotoxicology Program ~ Economic impact 17 • In the U. S., brain-relatetl dlcorders account for more howpitallaaflons than any other major ditease group. • Esttmated cost of tr•atm•nt, rehabilitation and r.tai.d consequences Is $ 400 blllton each year. Thtn, bra(nn-reiated toxiciy, rresults in longlWkry coats to htirnan h•aMh and the national economy. NCIR/FDA sr
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Dietert - 38 Johnson, B.E., R.G. Bell, and R.R. Dietert, Immunopharm. Immunotox. 12:237, 1990. Kaminski, N.E., and M.P. Holsapple. J. Immunol. 139:1804, 1987. Kammuller, M.E. et al., J. Toxicol. Clin. Toxicol. 26:157, 1988. Lang, D.S. et al., Fund. Appl. Toxicol. 21:535, 1993 . Luster, M.I., D.R. Germolec, and G.J. Rosenthal, Ann. Allergy 64:427, 1990. Luster, M.I., D.R. Germolec, G. Clark, G. Wiegand, and G.J. Rosenthal, J. Immunol. 140:928, 1988. Luster, M.I., A.E. Munson, P.T. Thomas, M.P. Holsapple, J.D. Fenters, K.L. White, Jr., L.D. Lauer, D.R. Germolec, G.J. Rosenthal, and J.H. Dean, Fund. Appl. Toxicol. 10:2, 1988. Luster, M.I., C. Portier, D.G. Pait, K.L. White, Jr., C. Gennings, A.E. Munson, and G.J. Rosenthal. Fund. Appl. Toxicol. 18:200, 1992. Lyte, M. and P.H. Bick, Int. J. Immunopharm. 8_:377, 1986. Marks, H.L., and R.D. Wyatt. Science 206, 1329, 1979. Myers, M.J., L.B. Schook, and P.H. Bick, J. Pharm. Exp. Ther. 242:399, 1987. Nadeau, D. and D.A. Lane, Cell Biol. Toxicol. 4:13, 1988. Nishikai, M., Y. Fundatsus, and M. Homma, Arch. Dermatol. I-LO:253, 1974. Olerup, O. and L. Emtestam. Immunogenetics 28, 310, 1988. Pevnitsky et al. Int. J. Immunopharmacol. 7:875, 1985. Raffray, M. and G.M. Cohen, Arch. Toxicol. 65:135, 1991. ao Roney, P.L., and A. Holian, Tox. App. Pharm. 100:132, 1989. C"I w CO Rosenthal, G.L. et al., Tox. Appl. Pharm. 107:555, 1991. ° Q'I ~10 n 0 i
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WS12 APOPTOSIS VERSUS NECROSIS Necrosis Irreversible cell injury; pathological event associated with cessation of synthetic function; inability to reverse mitochondrial dysfunction (ATP depletion); profound disturbance in membrane function and cell homeostasis. Inflammatory response is a hallmark of cell death by necrosis. Apoptosis A type of "programmed" cell death, i.e., gene-mediated; massive neuronal cell loss normally occurs during development; trophic factors play a roll (preventative); involves de novo gene expression and protein synthesis. Intranucleosomal DNA fragmentation is considered a hallmark of apoptosis. • APOPTOSIS Possible role in: Alzheimer's Disease Amyotrophic Lateral Sclerosis (ALS, Lou Gehrig's Disease) Huntington's Disease Parkinson's Disease Spinal Muscular Atrophy Transmissible Spongioform Encephalopathies (i.e., Bovine Spongioform Encephalopathy, or BSE; Mad Cow Disease) Oo CJ.i ch: ~ co ' © ~ ~
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WS10 0 100 G 20 10 2 4 • • 10 12 14 Days of treatments 0 Effect of ddC on Molt4F cell growth end the Intracellular content of ddCTP. Moft-4F cells were treated wNh 0 0.05(y, 0.1 (L+), and 0.2 (0) pM [3H)ddC (52 CUmmoq Chen and Cheng, 1989 0 I F1H F1 MPP" 0 I DHP 0 MPP • MPP4' redox chemistry a Amn.«r.1wz :o rAPe 00 w _ (.N cO 0 41~ 0 r.>
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WS3 I* Mechanism of Neurotoxicity William Slikker, Jr., Ph.D. Neurotoxicity may be defined as any adverse effect on the structure or function of the central and/or peripheral nervous system by a biological, chemical, or physical agent. Neurotoxic effects may be permanent or reversible, produced by neuropharmacological or neurodegenerative properties of a neurotoxicant, or the result of direct or indirect actions on the nervous system. A multidisciplinary approach is necessary to assess neurotoxicity because of the complexity and diverse functions of the nervous system. Many of the relevant effects can be measured directly by neurochemical, neurophysiological, and neuropathological techniques, whereas, others must be inferred from observed behavior. Three postulated mechanisms of neurotoxicity will be explored. First, the excitotoxic hypothesis will be described with a focus on domoic acid, a seafood derived neurotoxicant. Excitotoxicity refers to the mediation of neuronal death by glutamate and related excitatory amino acids. Excitotoxicity is primarily mediated by glutamate receptors with N-methyl-D-aspartate receptor activation more rapidly resulting in neuronal calcium influx and lethal injury than kainate or alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (ANPA) receptor activation. Second, the oxidative stress hypothesis of neurotoxicity will be addressed with the use of peroxides and metals as postulated causative agents. The role of free ~ radicals in the induction of oxidative damage to lipid membranes, enzymes and proteins of brain cells will be examined. Third, mitochondrial dysfunction will be discussed as a mechanism of both central as well as peripheral neuropathy. The central neurotoxicant 1-methyl-4-phenyl- 1,2,3,6,tetrahydropyridine (MPTP), for example, is postulated to induce toxicity by inactivation of NADN dehydrogenase and hence, mitochondrial respiration after its enzymatic activation to MPP`. The dideoxynucleoside, dideoxycitidine (ddC) is postulated to induce peripheral neuropathy by selectively inhibiting mitochondrial DNA synthesis after activation to its corresponding triphosphate (ddCTP). Finally, the possibility that multiple mechanisms may be involved with neurotoxicity will be discussed. CO w ~ C?J cO Cj L4 1~0 UI
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11110~a/ NAOHCEHlOPOOENAEE 14 r.t m." MPP+ t 0 © 0 eueceuTe cewomaENAet P hJ daMwq u lUGCNNTE nH+ I 40. ~ 0 HypolAetkW reaetion sXB of MPP* In the respUalory rhaln. srw.naa.mam voo nH* CYIOCI~E! OXDME r a4 .}o, ISO WS9 0 .
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Page 1 Protection Against Free Radical Damage Mary Treinen Moslen, Ph.D. William C. Levin Professor of Environmental Toxicology Director, Toxicology Training Program • University of Texas Medical Branch Galveston, Tezas Mary Treinen Moslen was bom in Iowa, received a BA in chemistry from Vassar College, a MS in scientific communication from Boston University, and a PhD from UTMB. Her research interests are cell injury and toxicant effects on bile formation. She is a member of the Society of Toxicology and the Society of Investigative Pathology. Her service on national committees included membership on the NIH Toxicology Study Section. She has served on the editorial boards of several journals and as an associate editor of Toxicology and Applied Pharmacology. 00 tN ~ Ctq tJ 41. co CD
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A multidisciplinary approach Is necessary to assess neurotoxicity because of the complex and diverse functions of the nervous system. Research Approach Evaluation of Available Endpoints Neuropathology Neurochemistry Neurophysiology Behavior li~ht microscopy transmitter levels EEG spontaneous histochemistry receptor binding evoked potentials (functional) electron enzyme activities single-unit schedule- microscopy recordings controlled "challehge" Neurotoxicity Profile LCH8£28 Neurotoxicology Program food/Contaminants Aesfiddes Metais Cycod Flour 2.4-D; 2,4,5-T Mercury Domotc Acid Orqanophosplwtes Lead Glutamate Carbamates Manganese iherapeulic Drugs Druys of Abuse FenBuramine MettwmpMtarnine Fludarabine/Taxol MDMA Anti-HIV Cocaine Postulated mechanisms of neurotoxicity • Excitotoxic hypothesis • Oxidative stress hypothesis • Mitochondriai Dysfunction C 9
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Page 3 0 PRIMARY AND AUXILIARY PROTECTION SYSTEMS Primary Protection Systems . Antioxidants Antioxidant Enzymes Vitamin E (a-Tocopherol) Superoxide Dismutase Enzymes (DOD) (3-Carotene Catalase Glutathione (GSH) Glutathione Peroxidase Vitamin C (Ascorbate) Glutathione Transferase Aldehyde Dehydrogenase Transition Metal Binders Transferrin Ferritin Ceruloplasmin Albumin Metallothionein Auxiliary Protection Systems Antioxidant Regenerators MISC GSSG Reductase Phospholipase Glucose-6-P-Dehydrogenase GSSG and GS-Conjugate Exporters co w Membrane Radical Reductase U4 00 C:) Ith • CD Modified from Moslen, 1992
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0 GLUTAMATE l INDUCTION I:TE CG No CI iP3 DAG OLUTA/AA AMPLIFlCAT1ON BWELLINO I p EXPRESSION DELAYED DEOENERATION PHYSIOLOGY 86208228 CIm1.1992 PATHOPHYSIOLOGY Zinkend et al,1992 Two Patterns of Excitotocity on Cortical Neurons R.c.ptor Exposura ilm. Rnpkilytrtypw~d NMDA 3-5 min ExcNOtoxIcNY Slowly Uipp.rid AMPA Hours Excllotoxlclly K alnat. H mutamlc acid y~<< 1 Kelntc actd 7 y .virlfL'AT ~ rn ?,} til Pc:i~ sfiLip1 0 Rate of Route of Ca" hdtuz Ca•• IMux Fost MNDA r.copta- patol charu»I Sbw ~roltaw9aw Ca 2' I TNA - Ca2'
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• MAO-B DopamUro . ~~ M~ (ilial Cells MPP Carrier Concentration WS8 Inhibitlon of NADH by mitochondria dehydrogenase ATP 0911 depletion death Dopaminerglc Neurons Steps in the expression of the neurotoxicity of MPTP Singer snd Ramssy,19Y0 0 lC'J N~ V~ /,t~' / ~ , (. f f.1i3 Sonsalla and Golbe, 1988 Concemratlonby Penetration into Cytoptaamk etectrochemlcaf hydorphobls (Hpp+ Gradient binding site Inhibition of (201o 30 µ)A) IntramltochondAal NAIRi dohydoryenase MPP+ (a1o mMl) . Step 1 Step 2 . Fate of MPP''after being pumped Into the neuron. Singer and Ramsay, 1990
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! Wrecking Crew of Reactive Oxygen Species (ROS) Superoxide anion radical, .Oz , can be considered as the master radical because it gives rise to other radicals. Hydrogen peroxide, H202, results from radicals and readily generates other radicals. H202 can cross membranes and begin the oxidative chain reactions that damage membranes. Page 5 Hydroxy radical, .OH, is considered the most reactive and dangerous ROS species because contact with •OH damages almost any biological molecule. Upid peroxy radical, LOO., represents the radical formed in lipid peroxidation reactions. LOO. has a surprisingly long half life of seven seconds. Co Cartoon slightly modified from Hooper (1989).
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Dopamine Concentration In Striatum (1 year old mice) 2000 1500 1000 5ao 0 Minutes after MPTP Dose (40mg/kg,ip) 'pa0.05 significantly different from control C 15 30 60 120 ROS in Striatum (1 year old mice) as 0.1 o.o C 15 30 60 120 Minutes after MPTP Dose (40mglkg,IP) •p.o.b5 significantly different from control wsii 0 ! 00 W W CO "J 0 -p O W
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Page 9 . > LIPID PEROXIDATION REACTIONS •OH ` I H ~III0 2 PEROXIDATION 00' w DETOXIFICATION REACTIONS RADICAL SCAVENGING 0 PHUSPHGLIPASE OOH GLUTATHIONE PEROXIDASE GLUTATHIGNE TRANSFERASE R or V GSH O HO~ ~~OOH u~, HO-Z SG z . ~ . CX> cm HOO? ~ HO-~ co R C:.J ~ ON
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D INITIATION PEROXIDATION IPROPAGATION DECOMPOSITION RADICAL SCAVENGING GLUTATRIONE PEROSIDASE ALDEHYDE DEHYDROGENASE GLOTATRIONE TR7INSFERASE D LIPID PEROXIDATION REACTIONS rage tu Polyunsaturated fatty acid of a membrane phospholipid is converted to lipid radical by an "9nitiation" reaction with • OH. The lipid radical is converted to a lipid peroxy radical (LO0 •) by a"peroxidation" reaction with 02, Note the shift of a double bond to form a conjugated diene. Lipid peroxy radicals are highly unstable species which will abstract a hydrogen from a nearby lipid species (LH) in a "propagation" reaction to form a lipid radical (L• ) plus a lipid hydroperoxide (LOOH). Alternatively the unstable lipid peroxy radical will rearrange to form unstable endoperoxides that break to form alkoxy radicals (LO •), and will eventually "decompose" to shortened oxidized phospholipids, small reactive aldehydes (eg, malondialdehyde) , and reactive alkenals (eg, 4-hydroxynonenal). Also Fe can stimulate the decomposition of lipid hydroperoxides. (7 DETO)OFICATION REACTIONS Vitamin E can terminate lipid peroxidation reactions by donating a hydrogen atom (H•) to a lipid radical in a "scavenging reaction. After the enzyme phospholipase cleaves phospholipid hydroperoxides to fatty acid hydroperoxides, this reactive species can be detoxified by the enzyme glutathione peroxidase to a fatty acid. alcohol. Several aldehyde dehydrogenase isozymes readily detoxify malondialdehyde and other short chain reactive aldehydes to more stable acids. Glutathione transferase can detoxify 4-hydroxynonenal and other hydroxyalkenals by a GSH conjugation reaction. • 0 !
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Page 6 How Are Reactive Oxygen Species Detoxified ? 02 P450 oxidase NADPH oxidase Respiratory chain enzymes Cytosolic enzymes Oxidase OH' H20 ' The reactive oxygen species •OZ and H202 are formed by reactions in the endoplasmic reticulum (ER), mitochondria (MIT), and peroxisomes of cells. The very reactive radical •OH Is formed by electron transfer to H202 with Fe'+ frequently acting as the electron donor. The Fe+++ formed is cycled back to Fe+* by •OZ . •02 is detoxified by the enzyme superoxide dismutase (SOD) many orders of magnitude faster than the spontaneous dismutation reaction. H202 is detoxified either by catalase or glutathione peroxidase with the oxidation of GSH to GSSG. The GSSG formed is regenerated back to GSH by ~ the enzyme glutathione reductase ~ 2GSH\ Glutathione Glutathione peroxidase reductase . '- GSSG'--' •OH Is extremely reactive and not readily detoxified. (Diagram from Robbins et al, 1989)
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Do Cells Within Tissues Contain Similar Amounts of Protection Systems ? . • Immunohistochemistry is a powerful tool for evaluating the distribution of protection systems in different populations of cells within a tissue. This procedure involves the use of antibodies to a given protein for the localization of that protein within a given tissue. Figures on this page compare the distribution of antibodies to the Mn-dependent form of SOD (anti-MnSOD) and to catalase (anti-CAT) in the kidney of hamsters. Note the staining of the antibodies to both MnSOD and catalase in the cells of the proximal tubule (PT), while neither antibody showed appreciable staining in cells of the glomerulus (G). Figures from Oberley et al, 1990.
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LVIOS(Gft Page 13 I* • GSSG Reductase GS-Conjugate ~ GS-Conjugate Exporter How Are GSSG and GS-Conjugates Exported ? Why Export GSSG and GS-Conjugates ? * GSSG can interact with protein thiols (RSH) to form mixed disulfides (R-S-SG). * GS-conjugates can inhibit both glutathione transferase and GSSG reductase enzymes. ADP GSH Transferase --o- GS-Conjugate ~ ~ ATP * ATP-dependent transporters move GSSG and GS-conjugates out of cells. * Hepatocytes preferentially transport GSSG and GS-conjugates into bile which is a direct route of hepatocyte excretion. c00,, W ~ ~ ID * Heart efficiently exports the GS-conjugate of ~ 4-hydroxynonenal, a toxic product of lipid oxidation. -
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Page 11 0 How Is Vitamin E Regenerated ? 1) By Ascorbate (Vitamin C) 2VtE 2 Vit E• • Ascorbate Dehydroascorbate 2) By membrane radical reductase 2 R• / ~ 2 vt E ~ membrane radical reductase 2 Vit E• 00 ~ U4 w c~» CD ~ ~ Cb
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Page 12 What Processes Use and Restore Glutathione ? • GLU, CYS, GLY 1 SYNTHESIS UTILIZATlON REGENERATION glutathione reductase ! r What Supplies the "Fuel" to Regenerate GSH ? Regeneration of GSH by G6PD GSSG NADPH glutathione glucose-6-P reductase dehydrogenase GSH ~ NADP t t GLUCOSE
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Page 8 What Processes Control ROS Formation by PhaQocvtes? B) Confinement Within a Phagolysosome . Ergutfinent : . , ~ P I YSOsome I -4° Attachment A_l N . ~. • ! . . . lormation and ,. degranutauon 0 • Cartoon of sequential events in the attachment, engulfment and phagocytosis of a particle by a phagocyte. Bacterial particle attachment stimulates activation of the NADPH oxidase that is localized to the phagolysosomal membrane. In addition, granules fuse with the phagocytic vacuole and release their contents into the phagolysosome. Contents of these granules include potentially destructive lysozyme, collagenase, elastase, phospholipase and myeloperoxidase, but also lactoferrin which binds up free Fe. This confinement within a phagolysosome limits, but does not total prevent, the escape of reactive oxygen species and other granule contents to the extracellular during phagocytosis. One type of powerful phacocytic cell, the neutrophil, is considered a "kamikaze" because it kills itself by apoptosis within 48 hr after attacking phagocytic particles. (Figure from Cotran et al, 1989)
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Page 14 EFFECTS OF VITAMIN E DEFICIENCY ON CCL4 HEPATOTOXICITY (Yoshikawa et a1,1982) Diet' Treatment Serum GPT= Liver TBA' (Karmen U) Reactants Vitamin E CCL4 250 50 ade uate Vitamin E CCL4 1200 375 1 2 3 Rats were fed diets either Vitamin E adequate (+) or Vitamin E deficient (-) for 4 months An index of liver damage (control value 20) An index of lipid peroxidation (control value 15) 0 0
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Page 18 Consequences of GSH Depletion in Erythrocytes ? 1) Erythrocytes have an impaired ability to detoxify reactive compounds, particularly peroxides. 2) Peroxides interact with membrane lipids and initiate destructive lipid peroxidation. 3) Peroxides interact with cell proteins, particularly with hemoglobin thiols (R-SH). Hemoglobin thiols are oxidized to intra- and inter-molecular disulfides. H202 + 2RSH mw-i• 2H20 + 2RS• RiS• + R2S• m--). R,S-SR2 4) The altered hemoglobins aggregate and form rigid Heinz bodies which limit the plasticity of the cells. Rigid erythrocytes are prematurely destroyed. ! ! Why Is G-6PD Deficiency So Prevalent ? * Highest incidence is in areas with endemic malaria. * Malaria parasites apparently depend upon erythrocytes GSH . * Low erythrocyte GSH impairs parasite survival. * Drugs which lower erythrocyte GSH maybe useful in the treatment of malaria. 0
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[Y[VJ[GTI Page 7 + What Processes Control ROS Formation by Phaeocytes? A) Regulated Activation of NADPH Oxidase Outside Membrane Inside \~ Fs ~ ae 2 91 ~ ropt GTP rapl ae 2 a 47 p, y NADPi GDP „ HADPM n -MV PP 260 kDa LATEIVT 0 P AC77VATED Hypothetical model of sequential events in the conversion of the latent subunits of NADPH oxidase to the active enzyme which reduces Oz to superoxide. Events in this activation include: a) assembly of cytosolic subunits, b) translocation of the assembled cytosolic subunits to the phagolysosomal membrane to form a complex with membrane subunits, c) polyphosphorylation of the 47-kDa subunit, and d) movement of the cytosolic rac 2 subunit to the membrane. This enzyme remains in its activated form for a limited duration. (Modified from Curnutte, 1993) 83380414
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1 v1VJlGlI Page 15 Consequences of Low Glutathione Peroxidase Activity ? Characterized in Vivo Due to Selenium Deficiency Glutathione Peroxidase Is A Selenium Dependent Enzyme Selenium in the Active Site LOCATION OF Se IN THE ACTIVE SITE OF GLUTATHIONE PEROXIDASE (Diagram from Ladenstein et al, 1981) co w w ~ co 0 4~- rD N
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1VlO5L6ft Page 17 • • What Is Glucose-6-Phosphate (G-6PD) Deficiency ? * A genetic defect which affects 100 million people. * G-6PD activity is essential in erythrocytes because of its role in the regeneration of GSH. * G-6PD deficiency causes short half-life of erythrocytes and vulnerability to drug-induced anemia. * Toxic drugs include primaquine sulfonamides and nitrofurantoin. * Erythrocyte injury in G-6PD deficiency is associated with: a) Decreased regeneration of GSSG to GSH b) Lower Vitamin E concentrations c) Oxidation of protein thiols * Treatment is Vitamin E to prevent thiol oxidation. Treatment of G-6PD Deficient Boys with Vitamin E [Hafez et al, J. Ped. 108:558, 1986] Controls Vit E (n=18) n=14 Before After Serum Vit E 0.95 0 50 1 5** (mg/dl) . . Reticulocyte <10 * Count (%) 3.0 2.3 RBC Half-Life 28 0 ** (day) . 16.9 22.8 * ** p<0.05, p<0.01 ~ I
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INTESTINAL LUMEN Page 27 0 BLOOD INTESTINAL LUMEN When Fe Excess 0 Increased Uptake from Blood © Increased Storage in Ferritin BLOOD
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Page 20 • Do Tissues Contain Similar Amounts of Protection Systems ? Glutathione In Tissues [Griffith and Meister, 1979] Liver 7.7 mM Kidney 4.1 mM Sm Intestine 2.9 mM Brain 2.1 mM Lung 1.5 mM Heart 1.4 mM Muscle 0.8 mM Plasma 0.028 mM Red Blood Cells 4.0 mM 0
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Page 19 • ! PROTECTION SYSTEMS IN PLASMA Action Actor transport or bind transferrin (Fe) Fe and/or Cu ceruloplasmin (Cu) albumin uric acid scavenge free glutathione radicals Vitamin C Vitamin E glucose 00 w w 40 cc ~ ~ rn
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Page 23 0 How Do Orcranisms Adapt to ROS Exposure? 1) Protocols which produce adaptation to high oxygen. a) Exposure to gradually increasing oxygen concentrations b) Challenge with a high oxygen concentration and then a brief rest before sustained exposure to high oxygen concentrations. For example, 48 hrs of 95% oxygen challenge and then a 24 hour rest. c) Lungs of adapted animals, which are the target tissues for oxygen toxicity, show elevated activities of one or more antioxidant enzymes. 2) Patterns of enzyme induction reported by Frank et al (1989) in the lungs of rats who were previously adapted to 95% oxygen by a 48 hr challenge and then a 24 hr rest protocol. ~ 300 SOD Catalose Glutathione Peroxidase 0 250 , P ~ 0.05 * c 200 . 0 U * ~ 150 C L 100 ~ CL 50 o 24 72 24 72 24 72 Hours of Reexposure to 95% 02 _ 3) Why are enzymes, as delicate molecules, such an important aspect of the adaptive response to ROS ? a) Antioxidant enzymes are inducible. b) As aptly stated by Harris (1992), "enzymes are designed specifically to execute reactions with speed, specificity, and high affinity" which are "desirable properties of any antioxidant m. 8338043D
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Major acute and chronic effects of Domoic Acid exposure Acul* Mtech Goslrointaflnal dbturbancos - nausea - vom7rynA - abdominal cramps - diarrhea or anoreda - N*urobplcal symptorm cnronIc .a.a. . In some onw a doewle add poba+tp. Pdn.nls rw.Yed u.a .Ne.~ef oe aMCUVw mwnmr Io.6 wUh p.mv.d o.~«a rr.N.cfted oe.nyand M«e mepaQ. n.ronon. n» m.mon d•nce Nqwn by UNw po1Nn1s b Mnoar to IM anM,.ac oApMwft a..ede.a by 3coville aed MM.r In 1958 In a pallwd who had uaerpp," we.ra...cwoi, a n» anrwdaluand MVaoewnpuL -headache - dirdness / loss of balance - rnemory Ioss Two types of histological lesions in the hippocampus - iype'A' leslon: Small focal ana of temlnal degen.rallon rstrict.d to tM CA2 *aMm Wcldurn. -lype 'B• Lslon: Extornl" Involvement of a larg.r ar*a of the hlppocampus. Including one or mor• regions of pyramidal neurons, and CAt and sublculum axon temdnals. Glht Irbrlttary acidic protNn (GFAP) slaining af one week rev.afed exbnNv. Alzhelmer's type 11 flllotb, primarily In the hippocampus of 1MN e1MmO1R 66rOH28 HYPOTHESIS Several diverse neurotoxic and neuropathological events lead to excess formation of free radicals or reactive oxygen species (ROS). These free radicals produce damage by oxidizing lipid membranes, enzymes and other proteins, and neuronal damage produced by these free radicals Is thought to play a major role in several neurodegenerative diseases. w 2 E o` 0 9
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Selenium Deficiency in Keshan Disease [Chinese Med. J. 92:471-476 & 477-482, 1979] ~ Endemic cardiomyopathy observed in a region of China, most susceptible group are children ~ Affected regions characterized by low Se levels of crops and human hair, and by low glutathione peroxidase activities of human blood ~ Se supplements beneficial to childrena n Morbidity rate from Keshan Diseaseb placebo controls 3,985 54 (13.5%) Se suppiementsc 4,510 10 ( 2.2%) children 1-9 years old in Mianning Country, Sichuan Province based on clinical signs, EKG and Xrays in 1974 0.5 - 1.0 mg once per week 0
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Why Do Nitroxides Have Theraneutic Potential? A) Family of stable free radicals compounds. R = H, TEMPO R = OH, TEMPOL R = CO, 4-oxo-TEMPO B) + •OZ" + 2H+ -~ + - O + HZOZ Page 24 0 0 I* Mimic activity of superoxide dismutase.
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Page 25 Why Do Nitroxides Have Therapeutic Potential? C. TEMPO Reported to Protect Isolated Perfused Hearts Against Detrimental Effects of Ischemia (5 min) Followed by Reperfusion 300 .~ m V 200 C 0 . 3 100 G 0 200 V 0 . C ontrol Ethanol TBNwp Treatment Conlrol 10 20 Reperfuslon time (min) g ventricular fibrillation IN ventricular tachycardia Q normal sinus rhythm ® other rhythm T 30 Data modified from Gelvan et al, 1991 on ra 600 Y r0 CJ ~ 500 ar N ~. R v ~ 00 on O ,_ 'O 300
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Page 28 What Is Wilson's Disease ? • Rare genetic disease of young people that affects I in 30,000. • • Characterized by neuropsychiatric changes related to degeneration of the cerebral basal ganglion, greenish-brown pigmented rings in the periphery of the cornea (Kayser-Fleisher rings), and liver damage. • Etiology is accumulation of Cu in tissues with very low biliary excretion of copper. • Genetic defect appears to be related to a gene product that transports Cu into bile. • Prognosis is progressive dementia and lethal liver cirrhosis - unless treated with a Cu chelator. 0
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MOStBl] Page 31 i HEMOCHROMATOSIS (Fe) or WILSON'S DISEASE (Cu) I Lipid Peroxidation of Organelle Membranes Direct Effect of Fe/Cu on Collagen Synthesis and/or .., Degradatio ~n Decreases in - Alterations in Fragility Mitochondrial Oxidative Microsomal Metabolism Enzymes, Cytochromes ! a ~ ~ Decreased Fe/Cu Excretion Hepatic Parenchymal Fe/Cu Overload ~ Cell Injury, Celt Death ~~ Fibrosis, Cirrhosis .--J I Hepatocellular Carcinoma Proposed pathophysiological mechanism of liver injury in chronic iron or copper overload. Note the progression from cell injury to fibrosis and then to neopiasia. Modified from Britton and Bacon, 1990. co w c.w ~ ~ t;u M
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Mosfen Page 21 s 9 Distribution of Protection Systems Within Cells ? Vitamin E and GSH B-Carotene Glutathione Transterase Glutathione Peroxidase Aldehyde Dehydrogenase ENDOPL ASMIC RETlCUL UM Catalase ( Vitamin E and GSH Cu/Zn SOD Glutathione Transferase Giutathlone Peroxidase Metallothionein PEROXISOMES LIPID BILAYER OF ALL CELLULAR MEMBRANES O LYSOSOMfS Vitamins C and E B-Carotene Vitamins C and E and GSH Cu/Zn SOD Glutathione Transferase Glutathione Peroxidase Aidehyde Dehydrogenase Ferritin Metallothloneln CYTOPLASM .E MlTOCNONDRlON Vitamin E D-Carotene Vitamin E and GSH Mn SOD Glutathione Transferase Glutathione Peroxidase Aidehyde Dehydrogenase Distribution of the antioxidants, detoxification enzymes, and transition metal binding proteins that comprise the intracellular protection system within cellular membranes and organelles. Note that the nucleus, with its vital genetic material, is protected by lipid and . water soluble antioxidants, by SOD, by glutathione transferase and peroxidase and by a metal binding protein. The mitochondrion, where •OZ is regularly produced by electron transport, is protected by multiple antioxidants, enzymes which detoxify •OZ , H202, and products of lipid peroxidation. (Modified from Moslen, 1992). Co w tr: ~ ~ ~ NJ Co
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Mos[ea Page 26 What Is Hemochromatosis ? * Common genetic disease of middle age that may affect 1 in 220 whites of northern European ancestry. * Characterized by hepatic fibrosis or cirrhosis, dilated cardiomyopathy, skin pigmentation, diabetes, arthritis, and testicular atrophy. * Etiology is accumulation of Fe in liver and then other tissues with diminished GI excretion from sloughed enterocytes. * Genetic defects appear to be related to the enterocyte gene products that regulate enterocyte uptake and/or storage of excess circulating iron. * Prognosis is good with early diagnosis and treatment by maintenance phlebotomies every 3-4 months. How Does the Body Control Fe Levels ? * Uptake of dietary Fe is regulated by the enterocytes of the of the intestinal mucosa. When body Fe levels are low, uptake of dietary iron is enhanced and less Fe is stored in the enterpcytes. * Plasma Fe is largeiy tightly bound to transferrin. * Transferrin-bound Fe enters cells by receptor-mediated endocytosis. * Within cells, Fe is incorporated into the many Fe-dependent proteins. Excess Fe is stored within ferritin molecules. With further accumulation, the excess Fe is stored as hemosiderin within lysosomes. * Free Fe levels are normally kept low by the combination of extra- and intra- cellular binding proteins. 0 0 * Systemic Fe levels can be lowered by processes regulated by enterocytes. Specifically, enterocytes can up regulate synthesis of both the serosal transferrin receptor and the Fe binding protein, ferritin, which would enhance systemic Fe uptake and storage. CO * Fe stored in enterocytes is "excreted' into the intestinal lumen when "aged" w enterocytes are extruded into the intestinal lumen. W,~ ~CX3 c.-no 4-- C~4 014
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Page 30 How Do the Transition Metals Fe and Cu Affect Lipid Peroxidation ? Fe2+ /Cu` Fe3+ /Cuz+ e- e- e- H~ 02 H+ t H202 H~ -•0H superoxide hydrogen hydroxyl radical peroxide radical LOOH + Fe2+ /CuI +--- 3w- Fe3+ /Cu2+ + OH + LO• LOOH + Fe3+ /Cu2+--Op- Fe2+ /CuI+ + H+ + LOO• . 0
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I riow Does the Body Control Cu Levefs T ~ 0 DIETARY COPPER 17 5 my/duy/ TISSUE COPPER PROTEINS •Sup.rox/d* d/imulos. 0 yyrot/noa. •Cylorhrome c o+ldar. ' .Lyrln* o.ldaa. URINE ( c 70yy/doy) MddiILe-CU 0 Page 29 Dietary Cu is absorbed and presented to the liver as a complex with ceruloplasmin or histidine. These Cu-complexes enter hepatocytes by receptor-mediated endocytosis or a"transporter". Hepatocytes store a small amount of Cu as a complex with metallothionine (MT) where the Cu is ` available for short term needs. Usually, excess amounts of Cu are efficiently _ excreted into bile via a Cu transporter that is located on the bile canaliculus. Hepatocytes store excess Cu in lysosomes; these organelles will excrete some of this stored Cu when they release their contents into bile. (Top diagram from Gollan, 1990) - 83380436
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Page 32 0 SUMMARY i) Protection against the injurious actions of free radicals is provided by a complimentary complex of * scavenging agents * detoxification enzymes * regenerating processes * export/excretion systems that act within * organelies * cells * extracefiular fluids * whole organism. 2) Injury can occur when components of these systems are * overwhelmed by large numbers of free radicals * inadequate due to dietary or other deficiencies * not present or not functional in genetic diseases. • 0
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INHALATION TOXICOLOGY
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T.l9P1 -3- III. Summary A. A proactive approach to the conduct of toxicological studies relevant to human hazard characterization is advocated. 1. Use of multiple approaches: in vitro cell and tissue studies including human material, in vivo laboratory animal studies, controlled exposure of people when ethically feasible, epidemiological investigations, and computer modeling. 2. Use of a strong exposure-dose-response orientation linked to concern for induction of disease. 3. Inclusion of exposure/doses relevant to likely human exposures. B. Need for increased attention to role of repair. C. Risk assessments are only as good as the information on which they are based. D. Scientists need to give increased attention to providing input for assessing human health risks at realistic levels of exposure and avoid inappropriate fear mongering. • 0
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• Lecture Outline MECHANISMS OF INHALATION TOXICOLOGY I. Introduction A. Cigarette smoking is dominant risk factor for pulmonary diseases including lung cancer. B. Public policy for control of chemicals has been driven by fear. C Scientiats have rontributed to atmosphere -f fear by focusing nn classification of chemicals: carcinogen, teratogen, neurotoxin, etc. D. "The dose makes the poison" is applicable to mechanistic research. 0 E. Risk assessment process can provide valuable input to help guide mechanistic research that will yield results that reduce the uncertainty in future assessments of risk. II. Case Studies Illustrating Role of Mechanistic Data A. Formaldehyde 1. Equivocal evidence for formaldehyde-causing cancer in humans. 2. Abundant evidence for high-level exposures causing nasal cancer in rats. 3. High levels of risk projected using air concentration of formaldehyde as exposure metric. 4. DNA-protein cross-links serve as marker of delivered dose. 5. Use of DNA-protein cross-link in rats and monkeys as "dose metrics" produces markedly lower estimates of human cancer risk. 6. Cell-killing and compensatory cell proliferation appears to have key role in nasal cancer induction in rats. 7. Cell population weighted measures of cell proliferation correlate well with observed nasal cancer incidence. 8. Advances in future likely to come from approaches that C° integrate data on delivered dose, mutagenesis, cell proliferation and ~ pathogenesis of lesions. 00 ~ o -~ ON RG h L
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RoE1 -2- ! 0 B. Diesel Exhaust and Carbon Black 1. Equivocal evidence from epidemiological studies of lung cancer. 2. Clean evidence of increased incidence of lung cancer in rats exposed to diesel exhaust. 3. High levels of exposure of rats to diesel exhaust results in interrelated changes. a. increased lung burden of particles b. impaired clearance c. inflammation d. lung cancer 4. Similar effects are observed with carbon black minimizing the likely role of absorbed hydrocarbons on diesel soot having a role in lung cancer induction in rats. 5. Increased frequency of mutations in lung epithelial cells at high exposure concentrations provides key link between lung burden and inflammation, and lung cancer. 6. Findings have significance for evaluating results of rat bioassays and other particulate material. C. Fiberglass 1. Epidemiological evidence of increased incidence of lung cancer and mesothelioma with exposure to several types of asbestos. 2. Exposure of rats to chrysotile and crocidolite asbestos causes lung cancer and mesothelioma validating use of rat bioassay. 3. High-level exposure of rats to refractory ceramic fibers produces lung cancer and mesothelioma in rats while glass fibers do not produce increased incidence of cancer. 4. Persistence of fibers in the lungs appears to be key factor in cancer induction. 5. Production methods can be altered to produce glass fibers with reduced durability in the lung. Co 6. Fibers with reduced durability are likely to be even safer. ~ 00 0 C) -r!. 4-~ k-.3
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Page 35 ` V.L. Kinnula, J.D. Crapo, K.O. Raivio. 'Biology of Disease: Generation and disposal of reactive ""***a/// oxygen metabolites in the lung' Lrrboratory Investigation 1995;73(1):3-19. G.R. Buettner. 'The pecking order of free radicals and antioxidants: Lipid peroxidation, a-Tocopherol, and ascorbate.' Archives of Biochemistry and Biophysics 1993;300(2):535-543. B. Halliwell, J.M.C. Gutteridge, C.E. Cross. 'Free radicals, antioxidants, and human disease: Where are we now?' J Lab Clin Med 1992;119(6):598-620. S. Soboll, S. Grundel, J. Harris, V. Kolb-Bachofen, B. Ketterer, H. Sies. 'The content of glutathione and glutatione S-transferases and the glutathione peroxidase activity in rat liver nuclei determined by a non-aqueous technique of cell fractionation.' Biochem J 1995;311:889-894. D. Gelvan, P. Saltman, S.R. Powell. 'Cardiac reperfusion damage prevented by a nitroxide free radical' Proc Natl Acad Sci 1991;88;4680-4684. B.R. Bacon, RS. Britton. The pathology of hepatic iron overload: A free radical-mediated process?' HepatoloSy 1990;11:127-137. E.D. Harris. Regulation of antioxidant enzymes.' FASEB 1992;6:2675-2683. L. Frank, J. Iqbal, M. Hass, D. Massaro. New "rest period" protocol for inducing tolerance to high ~ 02 exposure in adult rats.' Am J Physio11989;257:L226-L231. A.E. Holley, K.H. Cheeseman. Measuring free radical reactions in vivo.' British Medical Bulletin 1993;49(3):494-505. B. Halliwell. 'Commentary: Antioxidant characterization.' Pergamon 1995;49(10):1341-1348. CO W co C) ~ h.J
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• MECHANISMS OF INHALATION TOXICOLOGY Roger O. McClellan Chemical Industry Institute of Toxicology Research Triangle Park, NC 27709 The field of inhalation toxicology is concerned with both (a) the entry and subsequent disposition of agents entering via the respiratory tract, and (b) responses of the respiratory tract to toxic agents. As a basis for ~_r_derstanding the toxic effects of inhaled materials, it is crucial to understand (a) the physical and chemical properties of the toxic agent, and (b) the biology and pathobiology of the respiratory tract. The toxicity of a material is then best understood within the context of the interrelations among exposure-dose-response and consideration of the added dimensions of exposure and time. The ultimate practical use of inhalation toxicology is in understanding and assessing the human health risks of airborne materials. This requires that information is developed that is useful in assessing human health risks that are a very small fraction of the total risk occurring in the human population, and for likely exposure levels of human exposure frequently many times lower than can be readily studied ~ in experimental systems. Thus, the focus of inhalation toxicology is on issues in extrapolation, principally from laboratory animals to humans and from high to low levels of exposure and associated risk. This presentation uses three cases to illustrate how mechanistic information can be acquired and impact on the risk assessment process. Research with formaldehyde is used to illustrate the importance of delivered dose in extrapolating from laboratory animal species to humans and from high to low levels of exposure. Studies with diesel exhaust and carbon black are used to illustrate the importance of acquiring a mechanistic understanding of the pathogenesis of effects seen at high levels of exposure for understanding the human risks of exposure to these materials. Information from investigations with fiberglass are used to illustrate how an understanding of the pathogenesis of fiber-induced disease can help guide the development of new products. These three cases illustrate the importance of closely linking research, risk assessment, risk management and risk communication activities to address important societal issues. The ultimate credibility of risk-based decisions is dependent upon the availability of sound scientific 00 information used in the risk assessment process. W W cxa O .~a ~ pc 4`[ !'}
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ROGER O. McCLELLAN, D.V.M. serves as President of the Chemical Industry Institute of Toxicology, a position held since September 1988.The CIIT is supported by dues payments from some 40 ~ leading industrial firms and has a mission of creating an improved knowledge base for understanding and assessing the adverse effects of exposure to chemicals. Prior to his appointment as President of CUT, Dr. McClellan was Director of the Inhalation Toxicology Research Institute, and President and Chief Executive Officer of the Lovelace Biomedical and Environmental Research Institute, Albuquerque, New Mexico. He began his career with Lovelace in 1966, serving as Director, Fission Product Inhalation Program (1966-1973), and later as Vice President and Director, Inhalation Toxicology Research Institute, Lovelace Foundation for Medical Education and Research (1973-i976). During his 22 years with the Lovelace organization, he provided leadership for development of one of the world's leading research programs concerned with the toxic effects of airborne materials. Prior to joining the Lovelace organization, he was a scientist with the Medical Research Branch, Division of Biology and Medicine, U.S. Atomic Energy Commission, Washington, DC (1965-1966); and a senior scientist (1963-1964) and biological scientist (1959-1962), Biology Laboratory, Hanford Laboratories, General Electric Company, Richland, WA. He received his Doctor of Veterinary Medicine degree from Washington State University in 1960. Dr. McClellan has served in an advisory role to numerous public and private organizations. He is past ~ Chairman of the Clean Air Scientific Advisory Committee, Member of the Executive Committee, and Chairman, Research Strategies Advisory Committee, Science Advisory Board, U. S. Environmental Protection Agency; Member, National Council on Radiation Protection and Measurements; Member, Advisory Council for Center for Risk Management, Resources for the Future; a former Member, Health Research Committee, Health Effects Institute; and service on National Academy of Sciences/National Research Council Committee on Risk Assessment for Hazardous Air Pollutants, and as Chair of the Committee on Toxicology. He also serves as Adjunct Professor at Duke University, University of North Carolina - Chapel Hill, North Carolina State University, University of New Mexico, and Washington State University. He is active in the affairs of a number of professional organizations, including past service as President of the Society of Toxicology and the American Association for Aerosol Research. He serves in an editorial role for a number of journals, including service as Editor of CRC Critical Reviews in Toxicology. He is a diplomate of the American Board of Toxicology and the American Board of Veterinary Toxicology. Dr. McClellan's contributions have been recognized by receipt of a number of honors, including election to membership in the Institute of Medicine of the National Academy of Sciences. He has a long-standing interest in environmental and occupational health issues, especially those ~ involving risk assessment and air pollution, and in the management of multidisciplinary research organizations. He is a strong advocate of the need to integrate data from epidemiological, controlled clinical, laboratory animal and cell studies to assess human health risks of exposure to toxic materials. ZCU-t 3
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Page 33 ! • REFERENCES B.R. Bacon, R.S. Britton. The pathology of hepatic iron overload: A free radical-mediated process?' Hepalology 1989;11:127-137. A. Benidick, L.J. Machline, O. Scandurra. 'The antioxidant role of Vitamin C.' Adv Free Radicals Biol Med 1986;2:419 E. Beutler. 'Glucose-6-phosphate dehydrogenase deficiency.' N Eng J Med 1991;324:169-174. G.R Buettner. The pecking order of free radicals and antioxidants: lipid peroxidation, a-tocopherol, and ascorbate.'Arch Biochem Biophys 1993;300:535-543. L.Y. Chang, J.W. Slot, H.J. Geuze, J.D. Crapo. Molecular immunocytochemistry of the CuZn superoxide dismutase in rat hepatocytes.' J Cell Bio11988;107:2169-2179. H. Esterbauer, R.J. Schaur, H. Zollner. 'Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related alehydes.' Free Radicals in Biol Med 1991;11:81-128. J.L. Gollan. Copper metabolism, Wilson's disease, and hepatic copper toxicosis. IN: "Hepatology A Textbook of Liver Disease" D. Zakin, T.D. Boyer, eds. 2nd ed W.B. Saunders, Phildelphia, 1990; pp 1249-1272. O.W. Griffith, A. Meister. 'Glutathione: Interorgan translocation, turnover, and metabolism.' Proc Natl Acad Sci 1979;76:5606 M. Hafez, E.S. Amar, M. Zedan, H. Hammad, AH. Sorour, E.S.A EI-Desouky, N. Gamil. 'Improved erythrocyte survival with combined vitamin E and selenium therapy in children rvith glucose-6-phosphate dehydrogenase deficiency and mild chronic hemolysis.' J Pediatr 1986;108:558-561. C. Hooper. 'Free radicals: Research on biochemical bad boys comes of age.' J NIH Res 1989;1:102-106. T. Ishikawa, H. Esterbauer, H. Sies. Role of cardiac glutathione transferase and of the glutathione S-conjugate export system in biotransformation of 4-hydoxynonenal in the heart.' J Biol Chem 1986;261:1576 Keshan Disease Research group of the Chinese Academy of Medical Sciences, Beijing. 'Epidemiological studies on the etiologic relationship of selenium and Keshan disease.' Chinese Med J 1979;92:471-482. ~ R. Laenstein, O. Epp, R Huber, A Wendel. The structure pf glutathione peroxidase from bovine red blood cells. IN: Selenium in Biology and Medicine. AVA Publishing Co 1981; Westport, P33.
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Page 34 D.C. Liebler, K.L. Kaysen, T.A Kennedy. Redox cycles of Vitamin E. Hydrolysis and ascorbid acid dependent reduction of 8a-(alkydioxy)tocopherones.' Biochemistry 1989;28:9772 M. Lombard, A.B. Bomford, R.J. Polson, A.J. Bellingham, R. Williams. 'Differential expression of transferrin receptor in duodenal mucosa in iron overload: Evidence for a site-specific defect in genetic hemochromatosis.' Gastroenterology 1990;98:976-984. D.Y. Mitchell, D.R. Petersen. 'The oxidation of a-(3 unsaturated aldehydic products of lipid peroxidation by rat liver aldehyde dehydrogenases.' Toz Appl Pharmacol 1987;87:403-410, T.D. Oberley, L.W. Oberley, A.F. Slattery, L.J. Launchner, J.H. Elwell. 'Immunohistochemical localization of antioxidant enzymes in adult Syrian hamster tissues and during kidney development.' Am J Patho11990;13 7:199-214. M.T. Moslen; M.T. Moslen, C.V. Smith, editors. Free Radical Mechanisms of Tissue Injury. Baton Rouge: CRC Press, 1992; Protection against free radical-mediated tissue injury. p. 203-15. M.T. Moslen; D. Armstrong, editors.Free Radicals in Diagnostic Medicine: A Systems Approach to Laboratory Technologies, Clinical Correlations, and Antioxidant Therapy. New York: Plenum Press, 1994;Reactive oxygen species in normal physiology, cell injury and phagocytosis. p. 17-27, T. Ogihara, M. Miyake, N. Kawamura, H. Tamai, M. Kitagawa, M. Mino. 'Tocopherol concentrations of leukocytes in neonates' Ann N Y Acad Sci 1989;570:487 A. Pietrangelo, E. Rocchi, G. Casalgrandi, G. Rigo, A. Ferrari, M. Perini, E. Ventura, G. Cairo. 'Regulation of transferrin, tranferrin receptor and ferritin genes in human duodenum' Gastroenterology 1994;102:802-809. S.L. Robbins, RS. Cotran, V. Kumar. Robbins Pathologic Basis of Disease. 4th ed. Philadelphia: W B Saunders Co; 1989; W. Stemme], I3D. Reidel, C. Niederau, G. Strohmeyer. Pathogenesis of genetic haemochromatosis' Europ J Clfn Invest 1993;23:321-329. J.J.M. van den Berg, J.AF. Op den Kantp, B.R Lubin, F.A Kuypers. 'Conformational changes in oxidized phospholipids and their preferential hydrolysis by phospholipase A2: a monolayer study.' Biochemistry 1993;32:4962-4967. T. Yoshikawa, Y. Furukawa, M. Murakami, M. Takemura, M. Kondo. 'Effects of vitamin E on D-galactosanrine-induced or carbon tetrachloride-induced hepatotoxicity.' Digestion 1982;25:222 J. S. Boesch, C. Lee, R.G. Lindahl. 'Constitutive expression of class 3 aldehyde dehydrogenase in cultured rat comeal epithelium' The Journal of Biological Chemisiry 1996;271(9):5150-5157. • • 0
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such jaundice, challenge with mestranol provokes a relapse e. Erythromycin estolate E. Likely reasons for nonpredictable hepatotoxins 1. Allergic response 2. Biotransformation abnormality 3. Examining only a few animals (0.01 % which is 20,000 people in 200 million - requires 30,000 animals) F. Compounds producing hepatic cancer 1. Dimethylaminoazobenzene 2. Aflatoxin VI. DETECTION OF DRUG INDUCED LIVER INJURY A.. Histopathology B. Dye clearance 1. BSP 2. ICG 3. Bilirubin ~ VII. METHODS OF STUDYING LIVER INJURY A. Intact animals B. Isolated perfused liver C. In vitro with isolated hepatocytes 1. Freshly prepared 2. Primary culture K8 0 0 0
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Moslen - I Page 19 110 ! 0 Detecting Products Formed by ROS - 4 Trichrome stain Nitroso- tyrosine Nitrotyrosine antibody stain L Lumen of Artery A Atherosclerotic Plaque F Fibrous Cap * Hemorrhage Biol Chem Hoppe-Seyler 375: 81, 1994 Proteins containing a nitrotyrosine formed by peroxynitrate-mediated nitration of tyrosine can be detected by monoclonal and polyclonal antibodies which differentiate nitrotyrosine from other forms of modified tyrosines such as aminott, rosine or phosphotyrosine. Immunohistochemical staining with such antibodies to modified aminoacids reveals the location of the modified molecules in normal versus pathological regions of tissues, in specific types of cells within a tissue, and to some extent at the subcellular level, for example, a nuclear location. .
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Moslen - II Page 7 0 What Processes Control ROS Formation bv Phagocvtes? A) Regulated Activation of NADPH Oxidase 'j%~~ ~- ` J ' I v u Y r, y r Outside Membrane Inside LATENT ti ~ ac 2 81 ~ a GTP Pp P ACTIVA TED : Hypothetical model of sequential events in the conversion of the latent subunits of NADPH oxidase to the active enzyme which reduces Ol to superoxide. Events in this activation include: a) assembly of cytosolic subunits, b) transiocation of the assembled cytosolic subunits to the phagolysosomal membrane to form a complex with membrane subunits, c) polyphosphorylation of the 47-kDa subunit, and d) movement of the cytosolic rac 2 subunit to the membrane. This enzyme remains in its activated form for a limited duration. (Modified from Curnutte, 1993) $„BpU31
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Moslen - I Page 23 . Robbins SL, Cotran RS and Kumar V, Robbins Pathologic Basis of Disease. 4th ed. WB Saunders Co, Philidelphia, 1989. ~ Wiseman H and Halliwell B. Damage to DNA by reactive oxygen and nitrogen species: Role in inflammatory disease and progression to cancer. Biochem J 313:17-29, 1996. Longmire AW, Swift LL, Roberts LJ, Awad JA, Burk RF and Morrow JD. Effect of oxygen tension on the generation of Fi Isoprostanes and malondialdehyde in peroxidizing rat liver microsomes. Pergamon 47:.1173-1177, 1994. Beckmann JS, Ye YZ, Anderson PG, Chen J, Accavitti MA, Tarpey MM and White CR. Extensive nitration of protein tyrosines in human atherosclerosis detected by immunohistochemistry. Biol Chem 375:81-88, 1994. * Pryor WA and Squadrito GL. The chemistry of peroxynitrite: A product from the reaction of nitric oxide with superoxide. Amer JPhysial 268: 699-722, 1995. . W W W C'> ~ .tc;
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Free Radicals L Formation of Reactive Oxygen Species (ROS) A. B. Pathways Macromolecular Targets 1. Lipids 2. Proteins 3. DNA IL Consequences of ROS III. Oxidized Metaboiites of Nitrogen A. B. Formation Toxicity IV. Measurement of ROS and Products Mosien Page 2 0
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Production of ROS by Redox-Recycling Semiquinone Free Radical Production of ROS by Activated Neutrophils Mcsslea Page 6 CD O\ u
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1bIoslea - F. Page 20 > LIPID PEROXIDATION REACTIONS iNIT[ATIOi~t N>PEROXR}AT1ON * Fz- Isoprostane j _ ethane ~ pentane ~ AQ77 4' 1~~ ~ Products marked with a star can be detected in tissues, blood, urine or exhaled breath. u2 ao-yV0 o~ j Ho-2 ~~ <O*
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Moslen - II Page 15 What Is Glucose-6-Phosphate (G-6PD) Deficiency ? ! * A genetic defect which affects 100 million people. * G-6PD activity is essential in erythrocytes because of its role in the regeneration of GSH. * G-6PD deficiency causes short half-life of erythrocytes and vulnerability to drug-induced anemia. * Toxic drugs include primaquine sulfonamides and nitrofurantoin. * Erythrocyte injury in G-6PD deficiency is associated with: a) Decreased regeneration of GSSG to GSH b) Lower Vitamin E concentrations c) Oxidation of protein thiols * Treatment is Vitamin E to prevent thiol oxidation. Treatment of G~6PD Deficient Boys with Vitamin E [Hafez et al, J. Ped. 108:558, 1986] • Controls Vit E (n=18) n=14 Before After Serum Vit E 95 0 ** (mg/dI) . 0.50 1.5 Reticulocyte <10 * Count (%) 3.0 2.3 RBC Half-Life 28 0 ** (day) . 16.9 22.8 * ** p<0.05, p<0.01 Why Is G-6PD Deficiency So Prevalent ? * Highest incidence is in areas with endemic malaria. * Malaria parasites apparently depend upon erythrocytes GSH . * Low erythrocyte GSH impairs parasite survival. * Drugs which lower erythrocyte GSH maybe useful in the treatment of malaria.
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Moslen - I Page 21 i 0 Inflammation Oxygen toxicity Che 7 . / ~ / Activated oxygon species (02:, HZOz, OHq co v.i rsd ® ~ CD r.~ Radiation micals ~~~ 02 ~ sion injury
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Page ^_::. Reading List Buettner GR. The pecking order of free radicals and antioxidants: lipid peroxidation, a-tocopherol, and ascorbate. Arch $iochem Biophys 300:535-543, 1993. Esterbauer H, Schaur R and Zollner H. Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related alehydes. Free Radic in Biol Med 11:81-128, 1991. Hooper C. Free radicals: Research on biochemical bad boys comes of age. JNIHRes 1:102-106, 1989. Moslen MT, Smith CV, editors. Free Radical Mechanisms of Tissue Injury. Baton Rouge: CBC Press, 1992; Protection against free radical-mediated tissue injury, p 203-215. Moslen MT, Armstrong D, editors. Free Radicals in Diagnostic Medicine: A Systems Approach to Laboratory Technologies, Clinical Correlations, and Antioxidant Therapy. New York: Plenum Press, 1994; Reactive oxygen species in normal physiology, cell injury and phagocytosis. p 17-27. Kinnula VL, Crapo JD, Raivio KO. Biology of disease: Generation and disposal of reactive oxygen metabolites in the lung. Lab Invest 73:3-19, 1995. Buettner GR The pecking order of free radicals and antioxidants: Lipid peroxidation, a-tocopherol, and ascorbate. Arch ofBiochem andBiophys 300:535-543, 1993. Halliwell B, Gutteridge JMC, Cross CE. Free radicals, antioxidants, and human disease: Where are we now: JLab ClinMed 119:598-620, 1992. Holley AE, Chesseman KH. Measuring free radical reactions in vivo. British Medical Bulletin 49:494-505, 1993. Halliwell B. Commentary: Antioxidant characterization. Pergamon 49:1341-1348, 1995. Wiseman H, Halliwell B. Review Artical: Damage to DNA by reactive oxygen and nitrogen species: Role in inflammatory disease and progression to cancer. Biochem J313:17-29, 1996. Marrow JD, Hill KE, Burk RF, Nammour TM, Badr KF, Roberts II LI. A series of prostaglandin_ . FZ-like compounds are produced in vivo in humans by a non-cyclooxygenase, free radical-catalyzed mechanism. Proc Nafl Acad Sci 87:9383-9387, 1990.° Seekamp A, Mulligan MS, Till GO, Ward PA. Requirements for neutrophil products_ and L-arginine in ischemia-reperfusion injury. Amer J of Pathol 142:1217-1226, 1993. - ! 0 w w w Grisham MB. Reactive metabolites of oxygen and nitrogen in biology and medicine. RG LarPdes Company, 1992.
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Do Cells Within Tissues Contain Similar Amounts of Protection Systems ? ! • Moslen - II Page 19 Immunohistochemistry is a powerful tool for evaluating the distribution of protection systems in different populations of cells within a tissue. This procedure involves the use of antibodies to a given protein for the localization of that protein within a given tissue. Figures on this page compare the distribution of antibodies to the Mn-dependent form of SOD (anti-MnSOD) and to catalase (anti-CAT) in the kidney of hamsters. Note the staining of the antibodies to both MnSOD and catalase in the cells of the proximal tubule (PT), while neither antibody showed appreciable staining in cells of the glomerulus (G). Figures from Oberley et a1,1990. co W w ~ 00 CD c~s ~.~ fr~
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Moslen - II Page 1 • Protection Against Free Radical Damage , Mary Treinen Moslen, Ph.D. William C. Professor of Environmental Toxicology Director, Toxicology Training Program University of Texas Medical Branch Galveston, Texas 9 Mary Treinen Moslen was born in Iowa, received a BA in chemistry from Vassar College, a MS in scientific communication from Boston University, and a PhD from UTMB. Her research interests are cell injury and toxicant effects on bile formation. She is a member of the Society of Toxicology and the Society of Investigative Pathology. Her service on national committees included membership on the NIH Toxicology Study Section. She has served on the editorial boards of several journals and as an associate editor of Toxicology and Applied Pharmacology.
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MastCO Page 4 How Do Antioxidants Scavenge Free Radicals ? Reaction of Vitamin C (Ascorbate) + RW L-Ascorbic Acid Ascorbyl radical Reaction of Vitamin E (ex-Tocopftero!) R• + cc Y a-Tocophero! Dehydro-L- ascorbic acid fl 31 a-Tocopherolquinone + RM 0
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PROTECTION SYSTEMS IN PLASMA 0-Garotene a-Carotene Actor alhum3n - transferrin (Fe) ceruloplasmin (Cu) glutathione ascorhate tocopherols carotenes lycopene Effects of Vitamin E Supplement (140 mg ) on Plasma Antioxidants Action transport or bind Fe andtor Cu antioxidant Lycopene Nonsmokers Smokers Basal (°!o change ) Basal (°!% change } PtasmaVftaminC ltmotq 43.5:61Q.1 (-36)$ 27.4A: $.6 § (-36) $ RBC VrtaminE pmoilg hemoglobin 20.1:L S.I (+147)$ 19.7t3.7 (+41)$ I2SCMDA p~noUg hemoglob#n 133+74 (-36)$ 372t180 § (-73)$ § Effect o€Smotung at p <4.45 $ Effect of Vitamin E at p< p,g5 Mosien - a Page 16 0
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Moslen -- I Page 18 Detecting Products Formed by ROS - 3 _ Free Radical-Initiated Formation of F2 - Isoprostanes ~ICCzH ArachidonicAcid ~ ~ R• I[L/~-' RH C02H ON N E Smoking Status and F2 - Isoprostanes Levels g ~ 2000 ~ • nonsmokers ° 1600 0 smokers 0 E ° 1200 0 a O 00 CO'N . • ro S 800• O m 0 0 400 q o• ! « 0 • " ~ 0 150 300 450 c co,x ~ Plasma FZ-Isoprostanes(pmomiter) ~iN/ ~ o N N Engl J Med 332: 1198-1203, 1995 Fz-Isoprostanes co,x Comparative Changes in Peroxidation Products after CCl4 Treatment MDA (nmoles/g liver) F2 - Isoprostane (pmole/g liver) Controls 220 f 132 13 t 2 CC14 (1mUkg) 527 f 132 1040 f 160 Fold Increase 2.7 80.2 Values are means f SD for 5 rats Longmire et al Biochem Pharmacol 47:1173, 1994. 83380U19
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Effects of Peroxidation on a Membrane ? Mosien - f Page 14 0 Membrane surface proteins It Lipid ~~ S ( 1 I ~ Ph h l d osp rpi c s ~ Upid Transmembrane glycflproiein 0 n Fatty acid Mafondialdehyde released,--oxidadon from oxidized fatty acids ~45 J ~~ tl .~ Lipid-lipid $-C.H3 croSSllrkkirig Orderly arrangement of membrane phospholipids and proteins in a representative cell membrane (top diagram) is disrupted by free radical initiated lipid peroxidation reactions (bottom diagram). Alterations to phospholipids include shortened oxidized fatty acids and the release of small reactive aldehydes such as malondialdehyde which can crosslink lipids or proteins. Alterations to proteins include strand scissions and inappropriate disulfide crosslinks. (Diagrams from Robbins et at, 1989) I*
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Detection > LIPID PEROXIDATION REACTIONS PEROXIDATION 00' Moslen Page 20 0 *ethane 41-) penane t Products marked with a star *10 can be detected in tissues, blood, urine or exhaled breath.
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Mosien - II Page 17 0 Do Tissues Contain Similar Amounts of Protection Systems ? Glutathione In Tissues [Griffith and Meister, 1979] 0 Liver 7.7 mM Kidney 4.1 mM Sm Intestine 2.9 mM Brain 2.1 mM Lung 1.5 mM Heart 1.4 mM Muscle 0.8 mM Plasma 0.028 mM Red Blood Cells 4.0 mM exO w r7a ~ co c!) 0 .s
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Moslen Page 19 0 Detection Prostaglandin F2-like Products 7001, 600~ -O• O./ ~ i ./~iw•M 4S\ 500 o, zoor ® 100 c - CC1q + CC1q Data of Morrow et al, 1990
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Detection Colorimetric Thiobarbituric Acid Assay jTBA] H Electrochemical 8-oxo-deoxyguanosine H,N R=H,dR Moslen Page 18 !
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Maslen Page 3 Excitation and Reduction of Molecular Oxygen 0 Singlet oxygen j•0:0•} 0:0: } j H 02 +H //- H+ 02 e -0- 02 e o-- H202 e-.- 0 H• -~- H20 0:0•t •0:0 H:O:O:H •O:H H:O:H Oxygen Superoxide Hydrogen Hydroxyl Woter anion peroxide radicol 0 Superoxide Production by the Mitochondrial Electron Transport Chain NADH dehydrogenese Ubiquinone eyt e3
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GRIPPO I-I RECEPTOR-LIGANDS INTERACTIONS LOCK AND KEY BINDING AND FUNCTIONAL ACTIVITY 0
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Moslen - ii Page 14 I Consequences of Low Glutathione Peroxidase Acfivity? Observed in those with Selenium Deficiency Glutathione Peroxidase Requires Selenium in its Active Site Selenium Deficiency in Keshan Disease jChinese Med. J. 92:471-476 & 477-482, 1979] ~ Endemic cardomyopathy observed fin a region of China, most susceptible group are children ~ Affected regions characterized by low se tevets of crops and human hair, and by low gtutathione peroxidase activities of human blood * So supplements beneficial to childrena n Morbidity rate lrom Keshan Dtseaseb placebo controls 3,985 54 (13,5%) Se suppiementsc 4,510 10 (2.2°k) a children 1-9 years old in Mianning Country, Sichuan Province b based on clinical signs, EtCG and Xrays In 1974 c o.5 - 1.0 mg once per week i 0
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Moslen Page 16 Oxidized Metabolites of Nitrogen: Reactions H2 0 2NO' + OZ--} 2NO2• H2 0 2NO2 •-;. NZ 04 ---Iw- NZ O4 + N03 - R2-NH + NO2' .--~ R2-N-N=O + 0 2 • + NOONOO' +H+ N02 • + HO ONOOH
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GRIPPO 1-2 0 GLOBULAR REPRESENTATION OF A RETINOID BINDING PROTEIN co cl~j C~j 00 0 a co co
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GRIPPO 1-5 0 CLASSES OF RECEPTORS MEMBRANE RECEPTORS SOLUBLE NUCLEAR RECEPTORS
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0 &lfi8mm8tion Mustea Page 21
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GRIPPO 1-8 9 EXTRACELLULAR SPACE AGONIST-REGULATED ENZYMES - CHO CHO CHO• CHO CHO CHO SS NH2 SS CHO ss ss CHO SS sS CHO SS SS CHO SS S5 IcCHO u MEMBRANE - RRBRBRRRR ::~RARRBBRRR 888888888-' 888888888 THR 654 0 C02H ATP LIGAND•BINDING DOMAIN TYROSINE KINASE I KINASE DOMAIN J~ PROTEOLYTICALLY CYTOPLASM 1-1-~ SENSITIVE REGION EJCTFIACELLULAR DOMAIN TYR 1068 AUTO-PHOSPHORYLATION TYR 1148 TYR 1173 SITE DOMAIN PROTEIN KINASE-UKE DOMAIN TRANSMEMBRANE C"O (A W r GUANYLYL CYCLASE tn ~r tz:) ~ DOMAIN CATALYTIC DOMAIN
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GRIPPO 1-3 0 MICROENVIRONMENT OF UGAND-RECEPTOR INTERACTIONS 0
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GRIPPO I-11 NUCLEAR RECEPTORS RAR beta VITAMIN D3 THYROID ESTROGEN .;Y~~eT .. . . . . _ xf:....._ PROGESTERONE 448 427 456 GWcocOancOID .~~.....:,_~..._.. ~'vYv .;; 44 595 777 930 0 0 •
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GRIPPO 11-14 • SCATCHARD ANALYSIS N N N s N s N N co ~ ao ~ ~ ~ ~10
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GRIPPO 1-6 AGONIST-GATED ION CHANNELS ! i
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GRIPPO 11-17 . Pharmacological Relationships Agonist, Antagonist and Partial Agonist 100 ! ;R 0 I .1 i ~ I 10 100 test substance 1000 I 10000 i
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GRIPPO 11-16 TWO TYPES OF UCAt3D-RECEPTOR INTERACTIONS BI()CHEMICAL IC€SYQtf SE d-RECEP7DR NU BIOCHEMICAL RESPONSE T. ,a-RECEPTOR ANTAGONIST DOES NOT PRODUCE A RESPONSE BUT BINDS TO RECEPTOR AND SLDL'LS ACCESS OF AGONIST
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I-9 Molecular Mechanisms of Retinoid-Induced Teratogenesis ARoroaches 1) Characterize the retinoid-sensitive teratogenic window in mice. 2) Isolate and characterize retinoid-responsive genes during the sensitive period. 3) Characterize the teratogenic potential of retinoid- responsive gene products using transgenic animal models.
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GRIPPO 11-18 4) EFFECT OF A COMPETITIVE AGONIST ON RECEPTOR ACTIVITY 100 No antagonist + Competitive antagonist 0.1 9 8 7 -6 -5 -4 -3 LOG (CONCENTRATION OF AGONIST) co w w ~ Cb c3 ~ CD s.u
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0 . Ol II - 2 Stage-Deoenoent Effect of all-trans-Retinoic acid (60 mgrkg) in C57 BU6 Mice 1.5 2.5 3.5 4.5 5.5 6.5 7.5 Pregnant dams rece ved a single oral dose of retinoic acid. Litters were examined at gestation day 17.5 pc.
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! Pesticides: Metabolism and Mechanisms of Toxicity Ernest Hodgson Department of Toxicology North Carolina State University Raleigh NC 27695 • 1. Pesticides - Chemical and Use Classes 2. General Toxicology and Metabolism of Pesticides 3. Organochlorines - Metabolism and Mode of Action 4. Organophosphorus and Carbamate Pesticides - Metabolism and Mode of Action 5. New Classes of Pesticides - Metabolism and Mode of Action 6. Mechanisms of Toxic Interactions of Pesticides cO w w ~ CO CD ~ ~ c~
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Pesticides: Metabolism and Mechanisms of Toxicity 2. General Toxicology and Metabolism of Pesticides 00 04 CA e0 ~ CD N CD (S\
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/N`HCt~CH, N' N CI" `N'~INHCH(CH~z Atrazine Carbaryl Nlalathion ~~. QN OC5H5 S ~ ~ CI--C~ \CHCOOCH _ ~ ' CHCH.,1., "i' Fenvalerate + N-CH3 OH Paraquat s (CH3O)2PSCHCOOCzH5 CHzCOOCzN Warfarin O CHCI hCCH3 11 i C6H5 From: Hodgson, E. and Levi, P. E. A Text- book of Modern Toxicology, 2nd Edn. 1997 0 • i
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Mechanism of Nuclear Receptor Action RAR Response Element Transactivation Assay Gene Coding Region AAAA B-galactosidase Luciferase Chloramphenicol Acetyl Transferase I! 50408f~8 ~ 9 Measurable Enzyme Activity
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9 GRIPPO 111-21 NUCLEAR RECEPTOR RESPONSE ELEMENTS Response Element Promoter AGAACAnnnTGTTCT GRE GENE A AGGTCAnnnnnAGGTCA RARE ~a !~CUt`: af af'"' • I C7N
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Y-3 (CzHsO)zP-O-_~ .rN02 Parathion ~~ ~ ` 0 ~ P= Ci (CzlisO)zP-O Paraoxon 0 u _ HO--~ ~-NOz - (CzH50)zP0 p-Nitrophenol Diethyiphosphate . .,z , ,_, , ll~ S n . (CzH50)ZPO Diethyl nhnenhnrnfhinatf? From: Hodgson, E. and Levi, P. E. A Text- book of Modern Toxicology, 2nd Edn. 1997 r i 0
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• 9 Structure OCONHCH3 OCH(CH~z Name Cart aryl (Sevin) LD50 250-550 Propoxur(Baygon) 100 Aldicarb (Temik) -1 CH3 i CH3SCCH=NOCONHCH3 i CH3 From: Hodgson, E. and Levi, P. E. A Text- book of Modern Toxicology, 2nd Edn. 1997 Co W w CO 0 ~ CO
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aD Alddn Aflatoxin B1 TQ Dieldrin 0 O Afiatoxin B1 epoxide From: Hodgson, E. and Levi, P. E. A Text- book of Modern Toxicology, 2nd Edn. 1997 0 0
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D • . Pesticides: Metabolism and Mechanisms of Toxicity 3. Organochlorines - Metabolism and Mode of Action
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a DaT ci From: Hodgson, E. and Levi, P. E. A Text- book of Modern Toxicology, 2nd Edn. 1997 ! i ~~~-5 ~lD~' From: Hodgson, E. and Levi, P. E. A Text- book of Modern Toxicology, 2nd Edn. 1997
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! ! ~ Poaltlv• Alt.rpotentlal 10 meeC Figure 22-6. A schematic diagram of an oscilloscope record(n the depolarizarion and repolarization of a normal neuron (_c and one from a DDT-treated animal (- ----), showing the pruG gation of the negative afterporential (NAP). K:) ie From: Ecobichon, D. J. 1996. ti7 III:7
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0 Pesticides: Metabolism and Mechanisms of Toxicity 4. Organophosphorus and Carbamate Pesticides - Metabolism and Mode of Action • m W W • CO O N ;D r,.
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Table 22-6 Signs and Symptoms of Acute and Chronic To.eicih, Following Exposure to Organochlorine Insecticides INSECIICIDE CLASS ACUTE SIGNS CHRONIC SIGNS Dichlorodiphenviethanes DDT Parathesia (oral ingestion) Loss oi weight. anorexia DDD (Rothane) Ataxia. abnormal steooing Mild anemia DMC (Dimite) Dizziness. confusion. headache Tremors iJicofol (Kelthane) Nausea, vomiting Muscular weakness Methoxychlor Fatiflue, lethargy EEG pattern changes Methiochlor Tremor (peripheral) Hvperexcitabilitv. anxietv Chlorbenzvlate Nervous tension . Hexachlorocvclohexanes Lindane (y-isomer) Benzene hexachloride (mixed isomers) Cvclodienes Endrin Dizziness, headache Headache. dizziness, Telodrin Nausea, vomiting hvperexcirabilitv Isodrin Motor hyperexcitability Intermittent muscle twitchin_e Endosulfan Hyperret7exia and myoclanicjerking Heptachtor Myoclonicjerking Psvcholoeical disorders Aidrin General malaise includin; insomnia. Dieldrin Convulsive seizures anxietv, irritability Chlordane Generalized convulsions EEG pattern changes Toxaphene Loss of consciousness Epileptiform convulsions Chlordecone (Kepone) Chest pains. arthraigia Mirex Skin rashes Ataxia. incoordination, slurred speech. opsoclonus Visual difficulty, inability to focus and fixate Nervousness, irritability, depression Loss of recent memory Muscle weakness. tremors of hands Severe impairment of spermatogenesi From: Ecobichon, D. J. 1996. • 83380225
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Cyclodienes GABA \X CI if 0 Cyclodienes Figure 22-8. Proposed sites of action of cye/odiene-rvpe organo- chlorine insecricides on chloride ion transport by antagonizing GABA receprors in the chloride channel as well as Inhibition of Ca=•, Mg2+-ATPase. From: Ecobichon, D. J. 1996. 5--) ! !
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! 1C-z COO--(' "~CHzCH_-CHCH=CHz R H3C H 0 Pyrethrin I _~ Pyrethrin II (R=CH3) (R=COOCH3) CH(CHs)z i LD50 800-1500 CI--{\ /)-CHCAOCHC6HSOC6H5 CN li oI ' ~ From: Hodgson, E. and Levi, P. E. A Text- book of Modern Toxicology, 2nd Edn. 1997 M i
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Dietert - 34 THE CYTOKINE-IMMUNOGLOBULIN PATHWAY J!{4. • MffYCV"~' Plvlnn~,i- Environmental Factor -----> IL-4 ---> IgE -----> Atopic J Levels Levels Disorders Ir 11 00 crj w _ CO C:3 OW C~n rh
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i 0 i 0 SH NO2 CH3C0-, ~--NOZ T H,O -' CH,COH - HO-~ Z}NOZ From: Hodgson, E. and Levi, P. E. A Text- book of Modern Toxicology, 2nd Edn. 1997 tYO W CN ~ CO UJ -p A-Esterase n (CzH.°~ 8-Esterase 0 0 11 { + H20 -~ CH3CN2COH + CH3OH CH3CHZCOCHj O u CH3C5 O + Hz0 3- CH~COH + 0 0 CH30N~ + H_O -06~ CH,COH - C-Esterase
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Z 0 The CYP3A Subfamily • CYP3A5 OZPI - Expressed in the liver of 15% of the population - found in the placenta - substrate specificity similar to CYP3A4 • CYP3A7 - major fetal liver P450 - not found in adults except in placenta - metabolizes dehydroepiandrostene sulfate . CYP3A4 Activation • Activation- the metabolism of some substrates is increased in the presence of certain compounds • Autoactivation- Sigmoidal saturation kinetics • Mutagenic activity can be increased by CYP3A activators
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Dietert - 33 THE ANTIGEN PATHWAY Av Allergen -----> Antigen Presentation -----> Immune Response Environmental Host (Antigen Antigen Immune Factor + Product ---- > \Allergen --> Presentation --> Response , • i s
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Dietert - 30 0 VI. MECHANISMS OF AUTOIMMUNITY FACTORS IMPORTANT IN AUTOIMMUNITY' Genetic Factors HLA, Gm Allotypes, Target Organ Genetic Immune Factors Factors ~~,~y T'``) ,Y1°P' N-;?T Cell Defects, IgA Deficiency Hormonal Factors Sex Hormones, Thymic Hormones, Corticosteroids ~ Environmental Factors Drugs, Environmental Contaminants, Infections ` Shoenfeld, Y. and D.A. Isenberg, Immunol. Today 10:123, 1989. Numerous factors can influence and/or reflect the occurrence of autoimmune disease. 00 w 00 ~ w 0 (J4 co N)
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0 Effect of 7,8-Benzoflavone on Phenanthrene Metabolism • -4~ Effect of Phenanthrene on 7,8-Benzoflavone Metabolism ryi 0
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Dietert - TARGETED TOXICITY - AIDS Targeted Toxicity - AIDS No Infection HIV-mediated cytolysis is targeted tn (".D4 and Fusin Iiearing cells. il • i • Infoction ~ cytoiysis Infection . craP~ Viral Spread Iw
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Dietert - 39 Russell, G.I., R.F. Bing, J.A.G. Jones, H. Thurston, and J.D. Swales, Quart. * J. Med. 65#246:845, 1987. Shoenfeld, Y. and D.A. Isenberg, Immunol. Today 10:123, 1989. Sikorski et al., Fund. Appl. Toxicol. 13:843, 1989. Smialowicz, R. et al., Toxicology 64:109, 1990. Streilein, J.W. and P.R. Bergstresser. Immunogenetics 27:252, 1988. Tarloff et al. Toxicology 56:167, 1985. White, K.L., Jr., H.H. Lysy, J.A. McCoy et al., Toxicol. Appl. Pharm. 84:209, 1986. Yamashita, U. and T. Hamaoka, Gann 73:773, 1982. Zelikoff, J. et al., Fund. Appl. Toxicol. 22:1, 1994. • CO U11 tA CO C:) ~ Uj ~ _~
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