Toxicology for Industrial and Regulatory Scientists

Toxicology of Organ Systems

Mary Beth Genter, PhD, DABT, Fellow, ATSDepartment of Environmental Health

University of CincinnatiCincinnati, OH 45267-0056

(513) 558-6266( )Marybeth.genter@uc.edu

April 28 2015April 28, 2015

OverviewOverview

• For liver lung brain/nervous systemFor liver, lung, brain/nervous system, kidney, heart and skin: cell types, organ-related toxicants and methods of testingrelated toxicants, and methods of testing will be discussed

• For eye and gastrointestinal tract an• For eye and gastrointestinal tract, an abbreviated discussion of some relevant toxicants will be discussedtoxicants will be discussed

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Toxicology of the LiverToxicology of the Liver

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Drug-induced liver injuryg j y

• Most common reason to halt development of a drugdrug

• “Currently, no serum biomarkers, including the biochemical gold standard alanine gaminotransferase, can differentiate drug-induced from non-drug-related liver injury, can differentiate liver injury mediated by a specific j y y pdrug or mechanism, or can accurately predict the progression and outcome of hepatic injury”

– Ramaiah SK. Preclinical safety assessment: Current gaps, challenges, and approaches in identifying translatable biomarkers of drug-induced liver injury. Clin Lab Med 2011 31(1):161-72Lab Med. 2011 31(1):161-72.

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Cell types in the liver• Hepatocytes• Hepatocytes

– Majority of cells in the liver– Synthesize bile, contain metabolic enzymesSynthesize bile, contain metabolic enzymes

• Kupffer cells– Resident macrophages in liverp g– Ingest circulating particles

• Ito (stellate) cells( )– Main site of vitamin A storage in the body– Proposed to act as antigen presenting cells

A ti ti ( b th l) i d ti f– Activation (e.g. by ethanol) induces secretion of collagen, other extracellular matrix proteins

• Endothelial cellsEndothelial cells– Line sinusoids

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Liver - Structural Organization

Classic liver lobule consists of a portal tract (‘triad’)1) portal vein2) hepatic artery2) hepatic artery3) bile duct4) central vein)

• Hepatic blood flow and oxygen gradient are important factors affecting the metabolic activity of the liverthe liver

• Liver ‘zones’• Zone 1: Highest oxygen content; damage by direct acting agents;

efficient extraction of bile salts; highest level of glutathioneefficient extraction of bile salts; highest level of glutathione• Zone 2: Intermediate• Zone 3: ‘Hypoxic’; greatest concentration of cytochrome P450s

(CYPs)

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Organization of the liver

i idi idLiver lobule

CVCVsinusoidssinusoids

3

Blood drains from the portal tract toward the central vein

1

2

PVPVthe central vein

Bile flows toward the portal tract

HA BD

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CV

PT

Hata S, et al. Cytochrome 3A and 2E1 in human liver tissue: Individual

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2E1 in human liver tissue: Individual variations among normal Japanese subjects. Life Sci. 2010 86:393-401.

Courtesy of Prof. Memy H. Hassan

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Courtesy of Prof. Memy H. Hassan 10

Functions of the LiverFunction • “First pass” metabolism of absorbed materials

– Detoxification/degradationg– Bioactivation

• Detoxification of endogenous toxins– Bilirubin, ammonia

• Activation of testosterone– Administration of CYP inhibitors -> loss of secondary male sex

characteristics• Phagocytosis of materials such as bacterial fragments/endotoxin

– Kupffer cells• Synthesis of clotting factors, albumin, transport proteins (VLDL)• Synthesis and secretion of bile

– Many transporters are involved in this process• Dysfunction can occur without appreciable (histologically-evident) y pp ( g y )

cell damage11

Liver Cell Death(applies to other tissues, too)

• Necrosis (oncolytic cell death)– Cell swelling, leakage of cellular contents, nuclear disintegration

(karyolysis), and inflammatory cell infiltration( y y ) y– Generally affects many contiguous hepatocytes/parenchymal cells– Results in release of liver-specific enzymes into plasma (alanine [ALT]

or aspartate [AST] aminotransferases)– Histologically, inflammatory cell infiltrates and lack of nuclei and clear

cell membranes by H&E staining• Apoptosis (programmed cell death)p p (p g )

– A single-cell event that is programmed to eliminate aged or un-needed cells (e.g. during development)

– Associated with cell shrinkage; no inflammation– Caspases intracellularly cleave DNA and nuclear structural proteins

(apoptotic bodies)– Apoptotic bodies are phagocytosed by adjacent Kupffer cells or

h t thepatocytes• Hence, no release of intracellular contents

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Types of liver injury and representative agents

Fatty liver CCl4, ethanol, valproic acid, fialuridine, high fat diet

Hepatocyte death Acetaminophen allyl alcoholHepatocyte death Acetaminophen, allyl alcohol, copper, dimethylformamide

Immune-mediated response Halothane, diclofenacC li l h l t i Chl i tCanalicular cholestasis Chlorpromazine, estrogens,

manganese, cyclosporin A, 1,1-dichloroethylene

Bile duct damage Amoxicillin methylene diamineBile duct damage Amoxicillin, methylene diamine, sporidesmin, -naphthylisocyanate

Sinusoidal disorders Anabolic steroids, microcystin, cyclophosphamidey p p

Fibrosis, cirrhosis CCl4, ethanol, vitamin A, vinyl chloride, thioacetamide

Tumors Aflatoxin, androgens, arsenic, vinylTumors Aflatoxin, androgens, arsenic, vinyl chloride, hepatitis infection

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Fatty liver

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Assessment of Hepatic ToxicityHepatocyte necrosis• Hepatocyte necrosis– Histopathological evaluation– Clinical pathology assay for increases in liver enzyme activity (ALT)

• Bile duct damage• Bile duct damage– elevated alkaline phosphatase

• CholestasisJaundice increase in total bilirubin histological evaluation– Jaundice, increase in total bilirubin, histological evaluation

• Steatosis (fatty liver)– Histopathological evaluation (paraffin), Oil red O staining (frozen)

• Cirrhosis• Cirrhosis– Histopathological evaluation, special stains to detect fibrotic tissue

• Other potentially useful, emerging serum biomarkers:PON1– PON1

– Arginase 1– Glutamate dehydrogenase

Serum F protein– Serum F protein– Regucalcin

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LungLung

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Overview of the Respiratory tractOverview of the Respiratory tract

From Molecular and Biochemical Toxicology, RC Smart and E H d d 640From Molecular and Biochemical Toxicology, RC Smart and E H d d 640Hodgson, eds. p. 640.Hodgson, eds. p. 640.

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Function of the lung

• Function: Gas exchange– Dependent upon intimate contact between the very

thin processes of type 1 epithelial cells and blood endothelial cells in the alveolar walls

Micrograph of 4 alveoli (A) separated by C

C&D 7th, p. 612C&D 7th, p. 612

alveolar septum. C=capillaries19

Cell types in the lung

• Type 1: cells: gas exchange• Type 2 cellsyp

– Surfactant production– Division to repair lung injury

• Clara cells– Cytochrome P450-mediated bioactivation of compounds to toxic

metabolites in the lung happens most often in Clara cellmetabolites in the lung happens most often in Clara cell– Examples: naphthalene, styrene, 3-methylindole– Distribution of Clara cells varies greatly from species to species

• Pulmonary macrophages– Phagocytosis

C t ki l– Cytokine release20

Particle deposition in the respiratory t ttract

Directional change Air velocity

Very abrupt(impaction; 5-30 m)

++++

+++

Less abrupt(sedimentation; 1-5 m)

+++

++

Mild(diffusion; 1 m and less) --

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Toxicant Damage to the LungP th i f l d d b h i l• Pathogenesis of lung damage caused by chemicals– Bioactivation to reactive intermediates

• Clara cells > Type 2 cellsClara cells > Type 2 cells

– Free radicals, oxidative stress -ozone, NOx, tobacco smoke constituents

P ti l i ti b h– Particle ingestion by macrophages• Cytokine release, inflammatory cell recruitment

– Water solubility of gases define the pattern of toxicity of the y g p ygas

• Highly soluble gases (chlorine gas, formaldehyde sulfur dioxide, ammonia) do not penetrate further than the nose

• Highly insoluble gases (phosgene, nitrogen oxides, ozone) penetrate deeply into the lung responses

– Particles• Depending on particle size, they are deposited by interception

(fibers), impaction, sedimentation, and diffusion 22

CC10 immunohistochemistry of Clara cells in terminal bronchioles of mice

(A)Control mice shows intense and evenly distributed Clara cell-specific CC10 expression (black arrows).

(C) Consistent with Clara cell necrosis, a single dose of coumarin caused a significant diminution in CC10.

(E) Clara cell recovery as indicated by re-expression of CC10 (black arrow).

From Vassallo J et al. Food Chem Toxicol. 2010 48(6):1612-8Toxicol. 2010 48(6):1612 8

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Lung Injury

• Acute responses to injury– Pulmonary edema produces a thickening of the y p g

alveolar capillary barrier• Interferes with gas exchange at alveolar level

• Recovery is dependant on the severity of the initial injury

– Acute airway reactivity can be provoked by cholinergic drugs (acetylcholine) or other mediatorscholinergic drugs (acetylcholine) or other mediators (histamine, prostaglandins)

• Causes decrease in airway diameter and increase in resistance to air flow

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Lung Injury

• Chronic responses to lung injury– Fibrosis: fibrotic lungs contain increased amounts of g

collagen, and decreased surface area available for gas exchange.

– Emphysema: lungs become larger and too compliant, with destruction of the gas exchange surfaces

Asthma: caused by narrowing of the large conducting– Asthma: caused by narrowing of the large conducting airways

– Cancer: major causes are tobacco smoke; asbestosCancer: major causes are tobacco smoke; asbestos fibers; crystalline silica; metallic dust fumes (arsenic, beryllium, cadmium, chromium, nickel); and radon

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Phospholipidosis• Relatively common finding in drug discovery• Does not preclude clinical use of a drug• Does not preclude clinical use of a drug• Can be found in many organ systems in rodents

• Lung spleen heart brain kidney thymus gut eye• Lung, spleen, heart, brain, kidney, thymus, gut, eye…..• Most often manifests in the lung in humans• At light microscopic level cells appear to have• At light microscopic level, cells appear to have

inclusions (displaced nuclei, clear material in cytoplasm)y p )

• Electron microscopy is used as confirmation

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Drug-induced PLDDrug induced PLD

• May have pathological consequences orMay have pathological consequences, or not

• Many examples:• Many examples:– Amiodarone (anti-arrhythmic)

P h ili ( ti i )– Perhexiline (anti-angina)– Gentamicin (antibiotic)– Statin drugs (lipid lowering drugs)– Fluoxetine (antidepressant)

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Statin-treatment-induced PLD (light microscopy): Riu's stain smear of human bronchoalveolar lavage fluid shows a foamy appearance of the alveolar macrophages (arrows)

Amiodarone-induced PLD (transmission EM): Lamellated inclusion bodies (arrows) are seen in the l l h (AM) F M N t lfoamy appearance of the alveolar macrophages (arrows).

From Huang LK, et al. Statin-induced lung injury: diagnostic clue and outcome. Postgrad Med J. 2013 89(1047):14-9.

alveolar macrophages (AM). From Mesens N, et al. Phospholipidosis in rats treated with amiodarone: …….. supporting the lipid traffic jam hypothesis and the subsequent rise of the biomarker BMP. Toxicol Pathol. 2012 40(3):491-503. 28

Assessment of Lung Toxicity--humans

• Pulmonary function tests – Evaluate constrictive or obstructive airway changes

• Sputum analysis– Evaluate bacterial pathogens

B h l l L• Bronchoalveolar Lavage– Evaluate inflammatory cells, pathogens, extracellular

proteinprotein• Histopathological evaluations

– Tumors • Radiological exam

– Tumors, fibrotic lesions

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Example of pulmonary function testExample of pulmonary function test

Spirometry Some #s:Spirometry Some #s:• Total lung capacity=~6L• Residual volume=~ 1.2L• Vital capacity =~ 4.5L• Tidal volume =~ 0.5L

• FEV1 = amount of air that can be forcibly exhaled in one second (values range for ~3.5second (values range for 3.5 L for healthy adult females to ~5L for healthy adult males)

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C&D 6th edition Chapter 1530

Assessment of Lung Toxicity—lab animalsHi t l• Histology– “Inflation fixation” is critical– Morphometric reconstruction of airways– Morphometric reconstruction of airways

• Pulmonary function– plethysmograph p y g p

• Isolated perfused lung• Lung slices• Microdissection of certain cell populations• Pulmonary lavage

– Cell counts, protein content, etc.• Isolated lung cell populations

I l t d Cl ll– Isolated Clara cells ->31

Brain/Central Nervous SystemBrain/Central Nervous System

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Neurotoxicity• Any adverse effect in the structure or function of• Any adverse effect in the structure or function of

the nervous system: brain, spinal cord, peripheral nerves

• What is an adverse effect?– Hallucinations– Hallucinations– Convulsions– Ischemic injury (e.g. stroke)– Loss of sensory function (vision, touch, olfaction, taste, hearing)Loss of sensory function (vision, touch, olfaction, taste, hearing)– Hyperactivity/excessive nervousness– Decreased motor activity– Decreased I.Q./impaired learning– Memory loss– Headaches– Decreased motor function

L h– Lethargy33

Cell types in the nervous system

• Neurons• Glial cells

– Oligodendroglial cells• synthesize myelin in the central nervous system

S h ll– Schwann cells• synthesize myelin in the peripheral nervous system

– AstrocytesAstrocytes• form, together with brain endothelial cells, the blood-brain

barrier• ‘reactive gliosis’ in response to neuronal loss• reactive gliosis in response to neuronal loss

– Microglia• phagocytic cells in the nervous system

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NeuronsN th ll f th t th t d t• Neurons are the cells of the nervous system that conduct neurotransmission

• Communication with target cells occurs at the synapseCommunication with target cells occurs at the synapse• Two very important structural proteins

– Neurofilaments (3 subunits)– Tubulin (microtubules)

• Structurally, most neurons consist of – a nerve cell body

• Protein synthesis occurs only in the nerve cell bodyan a on– an axon

– one or more dendritesFrom Fishbach GD (1994) Scientific American Special From Fishbach GD (1994) Scientific American Special pReport “Mind and Brain”

pReport “Mind and Brain”

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AA

Nerve cell AxonAxoncell body

Myelin neurofilamentsmicrotubules

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Targets for neurotoxic damage

• Nerve cell body (neuron)A• Axon

• Myelin• Synapse

C&D 5th p. 465C&D 5th p. 465 37

Nerve cell body toxicantsOrgano mercury compounds• Organo-mercury compounds– E.g. methyl mercury—prenatal exposure can cause congenital brain

malformation• ManganeseManganese

– Toxic to neurons of substantia nigra—link to Parkinson’s?• Aluminum

– Once thought to cause Alzheimer’s diseaseg• Glutamate receptor agonists

– E.g. domoic acid• MPTP

– Heroin contaminant that caused Parkinson’s disease in addicts in the San Francisco area in the 1980s

– Mitochondrial toxicant, bioactivated by monoamine oxidases, substrate of dopamine transporterof dopamine transporter

• Noise, various solvents– Hearing loss due to damage to hair cells in the inner ear– Many solvents cause selective loss of hearing only at certain– Many solvents cause selective loss of hearing only at certain

frequencies

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Myelin toxicants• Hexachlorophene• Hexachlorophene

– Used in antimicrobial soaps• Triethyl tin

U d i i i t t t l l– Used in marine paints to prevent algal growth

• LeadA i d• Amiodarone– Potassium channel blocker, used as an

antiarrhythmic drugDi lfi• Disulfiram

Myelin toxicants cause ‘blebs’ in myelinCan result in complete loss of myelinClinical manifestation: slowed nerve

conduction velocityy

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Malik RA et al. Diabetologia. 2005 g48(3):578-85.

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Examples of axonal toxicants(many affect cytoskeletal proteins)(many affect cytoskeletal proteins)

• Taxol• Colchicine• Vincristine • n-Hexane • 2,5-Hexanedione• Carbon disulfide (CS2)• Acrylamide• Pyridinethione• Pyridinethione

C&D 5th p. 475C&D 5th p. 475

4141

Taxol and colchicine: microtubule t i ttoxicants

Stabilizes m’tubules

Inhibits polymerization of m’tubulesof m tubules

C&D 5th p. 477C&D 5th p. 477

4242

Crosslinking agents*

*crosslinking of neurofilaments in the axon causes axonal swellings and impairs a onal transportimpairs axonal transport

C&D 5th p. 474 C&D 5th p. 474

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Events at the Synapse

• Neurotransmission is initiated by the release of neurotransmitters into a synapse

E l f t itt i l d d i– Examples of neurotransmitters include dopamine, serotonin, acetylcholine

• After signal has been sent, neurotransmitter molecule must be removed from the synapse– Enzymatic degradation

Reuptake– ReuptakeFishbach GD (1994) Scientific American Special Report “Mind and Brain”Fishbach GD (1994) Scientific American Special Report “Mind and Brain”

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Toxic Events at the Synapse - examples

• Organophosphorous insecticides inhibit the enzyme acetylcholinesterase, which typically degrades the neurotransmitter acetylcholine, leading to prolonged signal transmission

T it hi l l d l ( li t )– Twitching muscles, excess glandular (salivary, tear) secretion

• Cocaine inhibits the dopamine transporterCocaine inhibits the dopamine transporter (DAT), which normally facilitates the re-uptake of dopamine from the synapse, into the pre-p y p psynaptic cell– Excess CNS dopamine receptor activation

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Evaluation of neurotoxicity• Histopathology• Histopathology

– H&E– Special stains: GFAP, Nissl, cresyl violet

• Teased nerve preparations– Nerve fibers are pulled apart with small forceps to examine

individual nerve fibersindividual nerve fibers• Demyelination, axonal swellings, inflammatory cell infiltration

• Electrophysiological testsE d ti l it– E.g. nerve conduction velocity

• Biochemical– E.g. cholinesterase inhibition (chickens)g c o este ase b t o (c c e s)

• Functional/behavioral tests– Functional observational battery (motor activity + histopathology)– Spatial memory– Anxiety/fear– Operant tasks

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KidneyKidney

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Introduction to Biochemical and Molecular toxicology, 3rd Ed, E Hodgson and RC Smart, Eds. P. 642.

48

Glomerulus

Prox convoluted tubule

Distal convoluted tubule

Cortical collecting tubuletubule

Cortex ↑

Cortical collecting tubule

Prox straight tubule

Medulla ↓

Medullary collecting

Descending limb

tubule

Descending limbof loop of Henle

Thin ascending limb of loop of

Papillary duct

limb of loop of Henle

Adapted from C&D 6th p. 49249

Kidney Functions

• Excretion• Extracellular fluid volume regulationg• Electrolyte homeostasis• Acid-base balance• Metabolism

– Vitamin D3 →1,25-dihydroxy vitamin D3, y y• Synthesis and release of hormones

– Renin– Erythropoietin

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Kidney - Susceptibility to Toxic Injury

• Renal blood flow is high compared to organ weight– Kidney ~1% of body weight but receives ~25% of

cardiac outputcardiac output• Blood flow to kidney is uneven

– Cortex receives highest blood flow and hence highest exposure to toxicantsexposure to toxicants

• Process of concentrating urine also concentrates toxicants– Toxicants can be concentrated by up to 200-fold– Precipitates can cause tubular obstruction

• Potential for bioactivation of toxicants– CYPs in proximal and distal tubules– Prostaglandin synthetase in medullary and papillary

cells

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Kidney – Site-specific Susceptibility• Glomerular toxicants

– Antigen-antibody complexes– Aminoglucoside antibiotics (gentamycin, tobramycin)– Adriamycin

• Proximal tubule:Proximal tubule:– Antibiotics (aminoglycosides, cephalosporins)– Antineoplastics– Radiographic contrast agents– Halogenated hydrocarbons (carbon tetrachloride chloroform tetrachloroethylene)Halogenated hydrocarbons (carbon tetrachloride, chloroform, tetrachloroethylene)– Heavy metals (mercury, uranium, cadmium, chromium)

• Distal tubule/collecting duct– Lithium– Tetracyclines– Tetracyclines– Fluoride ions– Amphotericin– Methoxyflurane

• Papilla• Papilla– Aspirin– Phenacetin– Acetaminophen

bromoethlamine– bromoethlamine– Other NSAIDs

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Kidney - Pathologic Responses• Acute renal failure

– Most common nephrotoxic damageCharacterized by abrupt decline in glomerular filtration rate– Characterized by abrupt decline in glomerular filtration rate (GFR) resulting in azotemia (high nitrogen content in the blood)

• Chronic renal failureP i d t i ti f l f ti ith l– Progressive deterioration of renal function may occur with long-term exposure to a variety of chemicals, including analgesics, lithium, and cyclosporine

• Adaptation following toxic insultAdaptation following toxic insult– The kidney can compensate for loss of renal functional mass– Following a nephrotoxic insult, cells that are non-lethally injured

may undergo cell repair/ adaptationmay undergo cell repair/ adaptation– Uninjured cells may undergo compensatory hypertrophy, cellular

adaptation, and proliferation, all contributing to the structural and functional recovery of the kidney

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Renal toxicity testingHi t th l• Histopathology

• Classical urinary markers of renal damage: albumin, total protein, glucose, pH

• More current (& sensitive?) biomarkers of renal toxicity– KIM-1: kidney injury molecule 1– Cystatin C: low MW protein; normally filtered by kidneyCystatin C: low MW protein; normally filtered by kidney– beta-2-microglobulin: low MW; normally filtered, then partially resorbed– NAG: β-N-acetylglycosaminidase activity– Clusterin: secreted glycoprotein synthesized in response to tubularClusterin: secreted glycoprotein synthesized in response to tubular

injury– Trefoil factor 3: one or more 38- or 39-amino acid domains in which 6

cysteine residues form 3 disulfide bonds to create a characteristicythree-leafed structure

– NGAL: neutrophil gelatinase-associated lipocalin

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Cardiovascular SystemCardiovascular System

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Anatomy of the HeartAnatomy of the Heart

www.bing.com/imageswww.bing.com/images56

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Cardiovascular System

• Function– Circulation of blood to supply tissues of the body with

nutrients respiratory gases hormones andnutrients, respiratory gases, hormones, and metabolites, while removing waste products of cellular metabolism and foreign matter

C ‘ ’• Cardiac muscle, like nerve cells, is an ‘excitable’ tissue

• Cells in the heart:• Cells in the heart:– Cardiac muscle cells = myocytes (25% of cells in heart)

• Myocyte contraction occurs as a result of an action potential causing release of calcium from the sarcoplasmic reticulum

– Cardiac fibroblasts (67% of cells in heart)– Other connective tissue cellsOther connective tissue cells– Purkinje cells– Vascular cells

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Cardiac ‘rhythm’

• Normally set by sinus P-node (pacemaker) cells• Arrhythmia: Deviation from the normal cardiac y

rhythm– Tachycardia: excessively fast heart rate– Bradycardia: slowed heart rate– Atrial fibrillation

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Electronic cell-to-cell coupling in the heart• Assessed by electrocardiography

– ECG

• Important deflections and intervals in an ECG• Important deflections and intervals in an ECG– PR interval: time from onset of atrial activation to the period of ventricular

activationQRS: conduction pathways through the ventricles– QRS: conduction pathways through the ventricles

– ST: interval during which the entire ventricular myocardium is depolarized

– QT: period of ‘electrical systole’; reflects the action potential duration– QT: period of electrical systole ; reflects the action potential duration• Prolonged QT interval is recognized as a life-threatening condition induced

by some drugs 59

Cardiac Toxic Responses

• Primary indicator of cardiac toxicity is decreased cardiac output, resulting in tissue hypoperfusion

N l d lt ti di t t i 5 L/ i– Normal adult resting cardiac output is 5 L/min• Cardiac hypertrophy

Associated with re expression of fetal genes– Associated with re-expression of fetal genes– Enlargement of existing cardiac myocytes

• Developing: cardiac workload exceeds output• Compensatory: cardiac output is maintained• Decompensatory: ventricular dilation develops and output

declines

• Heart failure– Inability of the heart to maintain sufficient cardiac output– Clinical analysis of right vs. left ventricular dysfunction

can predict prognosis 60

QT Prolongation and Sudden Cardiac Death

• Drug-induced QT-prolongation leads to ventricular arrhythmia and ‘Torsade de Pointes’ (TdP)Thi i id d di t i t• This is considered a severe cardiac toxic event

• Testing for QT prolongation is required by the U S FDA for drug developmentU.S. FDA for drug development

• Drugs associated with TdP:– Cyclooxygenase-2 (COX-2) inhibitorsCyclooxygenase 2 (COX 2) inhibitors

• Vioxx, Bextra– Antibacterials

• Fluoroquinolone• Fluoroquinolone– H1 receptor antagonists (Antihistamines)

• Terfenadine, astemizole

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http://www.youtube.com/watch?1 TO6fN bUv=1ccTO6fNobU

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Additional Cardiovascular System Toxicants

• Anthracyclines and other antineoplastic agents– Doxorubicin, 5-fluorocil, cyclophosphamide

CNS d• CNS drugs– Tricyclic antidepressants

• Ventricular arrhythmias, hypotension– Antipsychotic drugs

• Hypotension, ECG changes– General anesthetics

• Decrease myocyte contractility, arrhythmias, sensitization to other cardiac arrhythmogens

• Local anestheticsLocal anesthetics– Cocaine, procainamide

• extremely high doses cause slowed conduction speed, decreased rate of depolarizationdecreased rate of depolarization

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More Cardiovascular System ToxicantsAl h li b• Alcoholic beverages

• Heavy metals– Cobalt (‘beer drinkers cardiomyopathy’)– Cobalt ( beer drinkers cardiomyopathy )– Cadmium (some evidence)– Lead (hypertension)

I d t i l h i l• Industrial chemicals– carbon disulfide, styrene, 1,3-butadiene

Oil red O staining of lipid on aorticOil red O staining of lipid on aorticOil red O staining of lipid on aortic valves from mice exposed to

A. Control diet (C)B. HF diet (HF)C CS + C

Oil red O staining of lipid on aortic valves from mice exposed to

A. Control diet (C)B. HF diet (HF)C CS + CC. CS2 + CD. CS2 + HF

From Lewis JG Toxicol Sci 49:124-32, 1999

C. CS2 + CD. CS2 + HF

From Lewis JG Toxicol Sci 49:124-32, 1999

Assessment of Cardiac Toxicity• Abnormal heart rhythm• Abnormal heart rhythm

– Pulse rate determination• Tachycardia, bradycardia

• ECG analysis– AV Block

A i l i l fib ill i– Atrial, ventricular fibrillation– QT Prolongation (Torsade de pointes)

• Chest Pain (angina)• Chest Pain (angina)• Blood pressure changes

– Hypotensionypote s o– Hypertension

• Myocardial degeneration/necrosis– Myocardial degeneration/necrosis

• Histopathological examination 65

Biomarkers of Cardiac Toxicity• Creatine Kinase

– Multiple isoforms; CK-MB is considered a reasonably specific biomarker of acute myocardial infarction (MI)

M l bi• Myoglobin– Present in all muscle; serum levels increase dramatically 1-4 hr after

MIB type Natiuretic peptide• B-type Natiuretic peptide– Cardiac neurohormone; secreted in response to volume and

pressure overload; accepted as biomarker of congestive heart failure in Europe.p

• C-reactive protein– Marker of systemic and vascular inflammation; appears to predict

future cardiac events in asymptomatic individuals.• Cardiac troponins

– Cardiac troponin T (cTnT) and I (cTnI) are constituents of myofilaments; expressed exclusively in cardiac myocytesT t h b th ld t d d f di i f– cTn measurement has become the gold standard for diagnosis of

acute MI66

SkinSkin

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Four primary functions of skin

• Prevention of water lossP bilit b i t th i t• Permeability barrier to the environment

• Protection from invasion of microorganisms• Protection against abrasive action

• General structure:

EpidermisEpidermis

DermisDermis68

The Epidermis

Stratum corneum

Stratum compactum

Stratum granulosum

Stratum spinosum

Stratum basale

From Atlas of Normal Human Skin. W Montagna, AM Kligman, KS Carlisle, eds, 1992.From Atlas of Normal Human Skin. W Montagna, AM Kligman, KS Carlisle, eds, 1992.

6969

Epidermal Keratinocytes• Stratum corneum• Stratum corneum

– Outermost layer of the epidermis– Consists of several layers of completely flattened, keratinized cells

without cytoplasm or nucleiwithout cytoplasm or nuclei– Varies in thickness across the body, and by species– Resistant to water loss and pathogen invasion

• Stratum lucidumSt atu uc du– Found on parts of body with very thick skin and no hair (e.g. palmar

and plantar surfaces)• Stratum granulosum

– Irregularly-shaped cells containing profilaggrin– Release lamellar granules that release lipid into extracellular space

beneath the stratum cornium• These lipids including ceramides cholesterol fatty acids cholesterol• These lipids, including ceramides, cholesterol, fatty acids, cholesterol

esters, provide a barrier to chemical absorption across the skin• Stratum spinosum

– Several layers of irregularly shaped cells containing tonofilamentsSeveral layers of irregularly shaped cells containing tonofilaments– Connected by desmosomes to adjacent cell layers

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Epidermal Keratinocytes, con’t

• Stratum Basale– Single layer of cuboidal/columnar cells attached to the basement and to each other.– Undergoes continual mitosis

Self replacement of human (and pig) skin takes 30 days– Self replacement of human (and pig) skin takes ~30 days

BrdU labeling of stratum basale cells. Courtesy of Dr. Kevin Mills, The Procter and Gamble Co.

BrdU labeling of stratum basale cells. Courtesy of Dr. Kevin Mills, The Procter and Gamble Co.

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Epidermal Non-keratinocytes• Melanocytes

– Located within basal cell layer of epidermis,, hair follicles, sweat glands, sebaceous glandsg g

– Dendritic process intercalate between keratinocytes– Melanosomes move to tip of melanocyte process and are

phagocytized by adjacent keratinocytes• Merkel cells

– Associated with neurons in skin– Act as slow-adapting mechanoreceptors for touchAct as slow adapting mechanoreceptors for touch

• Langerhans cells– Found in upper stratum spinosum and have long dendritic

processes that extend into the granulosum cell layerprocesses that extend into the granulosum cell layer– Antigen presenting cells– Initiate some forms of immune-mediated dermatologic reactions

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The Dermis

• Forms the bulk of the skin• Composed primarily of filamentous, fibrous, andComposed primarily of filamentous, fibrous, and

amorphous connective tissue• Determines the tensile strength and elasticity of

the skin• Provides physical support for nerve and vascular

networksnetworks• Site of origin of skin appendages

– Hair folliclesHair follicles– Sebaceous glands– Sweat glands

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Toxic responses of the skin• Contact dermatitis

– Irritant dermatitis– Allergic contact dermatitisAllergic contact dermatitis

• Chemical burns• Granulomatous disease

T i ll f i b d ti l ti l i f t– Typically foreign body reactions; relatively infrequent• Phototoxicology

– Sunburn– Phototoxicity– Photoallergy

• ChloracneChloracne• Uticaria• Skin cancer

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Contact dermatitis

• Irritant dermatitis– Often cause damage/discomfort as a result of cumulative

irritationirritation• Soaps, detergents, solvents, cutting oils

– Can increase likelihood of allergic response by increasing t tipenetration

– Often accompanied by minimal inflammation• Allergic contact dermatitis• Allergic contact dermatitis

– Delayed (T-cell-mediated) response– Chemical penetrates through skin, binds to an

endogenous molecule, which is presented as an antigen by Langerhans cells to T-helper cells

• Examples: poison ivy (urushiol), 2,4-dinitrochlorobenzenep p y ( )

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Chemical burns

• Typically associated with strong acids or bases (alkalis)– Obvious examples: ammonia, calcium oxide, HCl, HF,

sodium hydroxide, toluene diisocyanate, hydrogen peroxideperoxide

– Not-so-obvious examples: ethylene oxide, methyl bromide

• Lesion is typically localized to site of exposure– One exception: HF can cause systemic hypocalcemia

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Phototoxicology

• Phototoxicity– Skin becomes red/blistered upon topical or systemic

exposure to certain chemicals and sunlightexposure to certain chemicals and sunlight– Chemicals causing phototoxicity most often absorb light

in the UVA range (320-400 nm)– Examples: Psoralens, PAHs, tetracyclines, eosin,

sulfonamides, sulfonylureas, thiazides, acridine orange• Photoallergy• Photoallergy

– Chemical elicits an allergic response by forming a complete antigen upon absorbing UV or visible light

– Examples: halogenated salicylanilides (antimicrobials), p-aminobenzoate (former sunscreen ingredient), some fragrances

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Chloracne

• Rare; occurs upon exposure to ligands for the aryl hydrocarbon receptor (AhR) and inducers of CYP1A1

• Skin lesions are precipitated by transition of b l d t k ti i i llsebaceous glands to keratinizing cells

• Chemicals: polychlorinated biphenyls/furans hth l /di inaphthalenes/dioxins• TCDD, 2,4,5-T

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Uticaria

• A.k.a hives• Elicited by allergens to which IgE antibodies y g g

have been elicited

Skin CancerSkin Cancer• Extensively studied in the ‘initiation-promotion’ y p

paradigm• Known causes in humans include radiation

(sunlight, X-rays), PAHs, arsenic

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Measuring skin toxicity/responses• In vitro

– Diffusion and Flow-through cells• Biopsy or cadaver skin is placed in a cell such that• Biopsy or cadaver skin is placed in a cell such that

a ‘donor’ fluid and flux of a test compound is monitored across the skin under static or moving fluid conditionsfluid conditions

– E.g. Bronough cells, Franz cells– 3-D skin models

• Skin fibroblasts differentiate into a fully-differentiated epidermis and respond to inflammatory cytokines.inflammatory cytokines.

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Diagram of a Franz diffusion cell

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Diagram of a Franz diffusion cell. From Karpanen TJ et al. Antimicrob Agents Chemother 2008 52:3633-36 81

Measuring skin toxicity/responsesMeasuring skin toxicity/responses

• In vivo– Patch tests—allergy testing– Percutaneous absorption and penetrationp p

• Species and body site considerations are important

– Transepidermal water loss (TEWL)– Histology (‘punch biopsies’)

Hi h f l d– High-frequency ultrasound– Fourier Transform Raman Spectroscopy

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Toxicology of the Gastrointestinal (GI) System

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The GI System• Extends from the mouth through the rectum; many

associated glands and structures• FunctionFunction

– Primarily to adsorb nutrients and water– One of the most important sites where toxicants are absorbed

• Transporter facilitated, as well as through and between epithelial cells

– Specialized transport systems facilitate the absorption of nutrients and electrolytes in the upper, middle, and lower small bowel

• No ‘specific’ transporters for toxicants• Xenobiotics can ‘hijack’ transporters for physiological substrates

and nutrients– E.g. the same transporter that facilitates calcium uptake also permits

lead transportlead transport

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Microscopic Anatomy of the Small IntestineMicroscopic Anatomy of the Small Intestine

Common structure throughout the gutCommon structure throughout the gut

VVMucosaMucosa

Villi: absorptionVilli: absorptionMucosaMucosa

Crypt: proliferationCrypt: proliferationMuscularis mucosaMuscularis mucosa

SubmucosaSubmucosa

Muscularis mucosaMuscularis mucosa

Circular muscleCircular muscle Wheater PR et al. Functional Histology (text), 1979 85

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Toxicology of the GI System

• Xenobiotics can affect many GI functions– Transit (GI motility, neurological interference)

– Absorption (transporter-mediated and paracellular transport)• Transporters: metal transporters, drug transporters, drug effluxors, etc.

Digestion (enzyme inhibition)– Digestion (enzyme inhibition)

– Secretion (ions, hormones, enzymes)

– Microflora• Depletion of microflora due to antibiotic administration

• Overgrowth causes altered coagulation due to bacterial secretion of vitamin K2K2

– Immunity (abundant submucosal lymphatic tissue; mucosal secretions)

– Pre-absorption biotransformation• ‘the grapefruit syndrome’: Furanocoumarins in grapefruit inhibit intestinal

CYP3A4, thereby increasing the amount absorbed of some drugs 86

Drugs exerting adverse effects on the GI tractDrugs exerting adverse effects on the GI tractConstipation Diarrhea Ileus Ulceration

Anticholinergics Antibiotics Loperamide NSAIDSAnticholinergics Antibiotics Loperamide NSAIDSAntihistamines Prostaglandins Tricyclics Corticosteroids

Diuretics Fatty acids Phenothiazines KClTricyclic

antidepressantsCholinergic

agonistsAnticholinergics Iron

preparationsIron

tiCholinesterase

i hibitOpiates

preparations inhibitors

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OcularOcular

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Ocular Toxicity - Anatomy of the Eye

Vit

Zonule Fibers

Vitreous Humor

Fovea

Aqueous Humor

Retinal Pigment

E ith li

http://www.webvision.med.utah.edu

Epithelium

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Compartments of the Visual System

• Eye– Cornea– Lens– Retina

R ti l i t ith li– Retinal pigment epithelium• Central visual pathway

O ti– Optic nerve– Optic tract

• Central processing areas• Central processing areas– Lateral geniculate nucleus– Visual cortexVisual cortex

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Ocular ToxicantsCornea Lens

(cataracts)Retina Optic nerve and

tract

UV radiation <310 nm

Corticosteroids Inorganic lead Acrylamide

Surfactants Sugars (diabetes mellitus)

Methanol Carbon disulfide

Organic solvents UV radiation Organic solvents Ethambutol

Acids (pH <2.5) Oxidative stress Organo-phosphates

B Ph hi i M dBases Phenothizazine drugs

Many drugsChemotherapeutics

Chloroquinetamoxifen 91

Assessment of Ocular Toxicity

• Ocular irritancy and toxicity– Traditionally based on Draize et al., 1944y

• In vivo assay, using albino rabbits; criticized because of pain to the animals, interlaboratory variability, poor predictive value for human irritantshuman irritants

– Alternatives assays are under development/consideration • Interagency Coordinating Committee on the Validation of g y g

Alternative Methods (ICCVAM)

• Some alternatives include use of isolated rabbit, chicken, or bovine eyes; hen egg chorioallantoic membranebovine eyes; hen egg chorioallantoic membrane

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Assessment of Ocular Toxicity• Clinical signs of ocular changes

– Jaundice, conjunctivitis

• Direct or indirect ophthalmoscopy• Direct or indirect ophthalmoscopy– Specialized expertise is required! Detects cataracts, retinal changes

• Electrophysiological techniques– Flash-evoked electroretinogram

• Electrodes record from the cornea after a light stimulus– Visual-evoked potentialsVisual evoked potentials

• Electrodes record from the visual cortex after stimulation– Electro-oculogram

• Used to assess retinal pigment epithelium status and eye movements• Used to assess retinal pigment epithelium status and eye movements

• Behavioral and Psychophysical methods– Assessment of whether stimuli can be discriminated or detected

• E.g. contrast sensitivity, luminescence threshold; visual acuity, color discrimination

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Resources(aside from the usual well-respected toxicology textbooks)

• Thoolen B et al. Proliferative and nonproliferative lesions of the rat and mouse hepatobiliary system. Toxicol Pathol 2010 38:5S.

• W Montagna, AM Kligman, KS Carlisle, eds, Atlas of Normal Human Skin. 1992, 2010.Costa DL Kodavanti UP Toxic responses of the lung to inhaled pollutants: benefits and• Costa DL, Kodavanti UP. Toxic responses of the lung to inhaled pollutants: benefits and limitations of lung-disease models. Toxicol Lett 2003 140-141:195-203. Review.

• Delaunois LM. Mechanisms in pulmonary toxicology. Clin Chest Med 2004 25(1):1-14. Review.

• Bolon B and Butt MT, eds. Fundamental Neuropathology for Pathologists and Toxicologists. 2011.

• Spencer PS and Schaumberg HH, eds. Experimental and Clinical Neurotoxicology. 2000.000

• Fuchs TC, Hewitt P. Preclinical perspective of urinary biomarkers for the detection of nephrotoxicity: what we know and what we need to know. Biomark Med. 2011 5(6):763-79.

• Waring WS Moonie A Earlier recognition of nephrotoxicity using novel biomarkers of• Waring WS, Moonie A. Earlier recognition of nephrotoxicity using novel biomarkers of acute kidney injury. Clin Toxicol (Phila). 2011 49(8):720-8. Review.

• Brock WJ, Somps CJ, Torti V, Render JA, Jamison J, Rivera MI. Ocular toxicity assessment from systemically administered xenobiotics: considerations in drug d l t I t J T i l 2013 32(3) 171 88development. Int J Toxicol. 2013 32(3):171-88.

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